Modified oligonucleotides, their preparation and their use

ABSTRACT

Modified oligonucleotides which possess at least one substituted 7-deazapurine base form more stable hybridization complexes with nucleic acids than unsubstituted analogs. They are useful as inhibitors of gene expression, as probes for detecting nucleic acids, as aids in molecular biology and as pharmaceuticals or diagnostic agents. Processes for preparing them are provided.

The instant application is a divisional of U.S. Ser. No. 09/643,233,filed Aug. 22, 2000 now U.S. Pat. No. 6,479,651, which is a continuationof U.S. Ser. No. 09/144,112, filed Aug. 31, 1998, now U.S. Pat. No.6,150,510, issued Nov. 21, 2000, which is a continuation of U.S. Ser.No. 08/554,164, filed Nov. 6, 1995, now U.S. Pat. No. 5,844,106, issuedDec. 1, 1998.

BACKGROUND OF THE INVENTION

The present invention relates to novel oligonucleotides which containmodified bases and which possess valuable physical, biological andpharmacological properties, to a process for their preparation and totheir use as inhibitors of gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for detecting nucleic acids, as aids in molecular biology andas pharmaceuticals or diagnostic agents.

Numerous chemical modifications of oligonucleotides are known from theliterature. These modifications can affect the sugar-phosphate skeletonor the nucleotide bases. Reviews of the state of the art are provided,for example, by Uhlmann & Peyman, Chem. Rev. 1990, 90, 543 and Milliganet al., J. Med. Chem. 1993, 36, 1923.

As a rule, it is necessary to modify oligonucleotides chemically sinceunmodified oligonucleotides are very rapidly degraded by nucleolyticactivities both in cells and in the cell culture medium. Stabilizationagainst nucleolytic degradation can be achieved by replacing thesugar-phosphate backbone or by modifying the phosphate bridge, the sugarmoiety or the nucleotide base [Milligan et al., above and Uhlmann &Peyman, above].

In addition to modifications which lead to oligonucleotides whichpossess increased stability towards nucleolytic degradation, thosemodifications are also of interest which alter the hybridizationbehavior of the modified oligonucleotides such that the latter are able,for example, to form more stable hybridization complexes (duplexes) withintracellular nucleic acids (so-called target nucleic acids). It ispossible to alter the hybridization properties of oligonucleotides by,for example, modifying the bases. The altered hybridization propertiesof oligonucleotides which have been modified in this way can berecognized, for example, from the melting temperatures (T_(m) values) ofthe duplexes, which temperatures are different from those obtained withthe unmodified oligonucleotides.

Thus, oligonucleotides which contain 5-bromouracil, for example, formmore stable hybridization complexes with the complementary nucleic acidsthan do oligonucleotides which contain the corresponding, unsubstitutedbases (uracil) [G. D. Fasman, CRC Handbook of Biochemistry and MolecularBiology, 3rd edition, 1975, Nucleic Acids, Vol. I , 58-585].

In addition, PCT Application WO 93/10820 discloses oligonucleotideswhich contain modified uracil or cytosine bases and which are able toform duplex or triplex structures with the target nucleic acids whichare more stable than those formed by the unmodified oligonucleotides.Oligonucleotides which contain the base analog 2-aminoadenine have alsobeen reported to exhibit improved hybridization properties [Chollet etal., (1988), Nucleic Acid Research, 16, 305-317]. German PatentApplication P4415370.8 discloses that incorporating 8-azapurine basesinto oligonucleotides increases the stability of the correspondinghybridization complexes with the target nucleic acids. In addition WO93/09127 discloses oligonucleotides which contain substituted orunsubstituted 7-deazapurine bases and which, as a result, are morereadily able to form triplex structures with the target molecules(double-stranded DNA). Oligonucleotides which contain 7-deazapurinebases which are substituted in the 7 position are also disclosed in WO94/22892 and WO 94/24144.

However, it is not possible to predict the base modifications which willlead to an increase in duplex stability. Thus, numerous examples areknown of base modifications which diminish duplex stability. Thus, PCTApplication WO 92/002258 describes pyrimidine-modified oligonucleotideswhich exhibit decreased binding affinity for the target nucleic acids.Methyl or bromine substituents which are introduced at the 8 position ofthe purine ring also decrease the stability of the correspondingduplexes [E. N. Kanaya et al., Biochemistry (1987) 26 7159, andBiochemistry, 1984, 23, 4219]. Oligonucleotides which contain7-deazaadenine form significantly fewer stable duplexes withcomplementary oligonucleotides than do oligonucleotides which containadenine [Seela et al., Nucleic Acid Research (1982) 10, 1389].

SUMMARY OF THE INVENTION

The object of the present invention is, therefore, to make availablenovel oligonucleotides which possess advantageous properties.

It has now been found, surprisingly, that oligonucleotides which possessat least one substituted 7-deazapurine base form hybridization complexeswith the target nucleic acids which are significantly more stable thanthose formed by comparable oligonucleotides which possess unsubstituted7-deazapurine bases.

The invention consequently relates to oligonucleotides of the formula I

and the physiologically tolerated salts thereof, in which

-   -   B are, independently of each other, a base which is customary in        nucleotide chemistry, and at least one B is a base of the        formula II        in which    -   R¹⁵ and R¹⁶ are, independently of each other,        -   1. hydrogen,        -   2. halogen,        -   3. (C₁-C₁₀)-alkyl,        -   4. (C₂-C₁₀)-alkenyl,        -   5. (C₂-C₁₀)-alkynyl,        -   6. NO₂,        -   7. NH₂,        -   8. cyano,        -   9. —S—(C₁-C₆)-alkyl,        -   10. (C₁-C₆)-alkoxy,        -   11. (C₆-C₂₀)-aryloxy,        -   12. SiH₃,        -   14. a radical as described under 3., 4. or 5. which is            substituted by one or more radicals from the group SH,            S—(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —NR(c)R(d), —CO—R(b),            —NH—CO—NR(c)R(d), —NR(c)R(g), —NR(e)R(f) or —NR(e)R(g), or            by a polyalkyleneglycol radical of the formula            -   —[O—(CH₂)_(r)]_(s)—NR(c)R(d), where r and s are,                independently of each other, an integer between 1 and                18, preferably between 1 and 6, with it being possible                for functional groups such as OH, SH, —CO—R(b),                —NH—CO—NR(c)R(d), —NR(c)R(d), —NR(e)R(f), —NR(e)R(g) or                —NR(c)R(g) additionally to be linked to one or more                groups, where appropriate via a further linker, which                favor intracellular uptake or serve for labeling a DNA                or RNA probe, or, when the oligonucleotide analog                hybridizes to the target nucleic acid, attack the latter                while binding, cross-linking or cleaving, or        -   15. a radical as defined under 3., 4. or 5. in which from            one to all the H atoms are substituted by halogen,            preferably fluorine;        -   R(a) is OH, (C₁-C₆)-alkoxy, (C₆-C₂₀)-aryloxy, NH₂ or NH-T,            where T is an alkylcarboxyl group or alkylamino group which            is linked to one or more groups, where appropriate via a            further linker, which favor intracellular uptake or serve            for labeling a DNA or RNA probe or, when the oligonucleotide            analog hybridizes to the target nucleic acid, attack the            latter while binding, cross-linking or cleaving,        -   R(b) is hydroxyl, (C₁-C₆)-alkoxy or —NR(c)R(d),        -   R(c) and R(d) are, independently of each other, R or            (C₁-C₆)-alkyl which is unsubstituted or substituted by            —NR(e)R(f) or —NR(e)R(g),        -   R(e) and R(f) are, independently of each other, H or            (C₁-C₆)-alkyl,        -   R(g) is (C₁-C₆)-alkyl-COOH; with the proviso that R¹⁵ and            R¹⁶ cannot each simultaneously be hydrogen, NO₂, NH₂, cyano            or SiH₃;    -   E and F are, independently of each other, H, OH or NH₂,    -   R¹ is hydrogen, C₁-C₁₈-alkyl, C₂-C₁₈-alkenyl, C₂-C₁₈-alkynyl,        C₂-C₁₈-alkylcarbonyl, C₃-C₁₉-alkenylcarbonyl,        C₃-C₁₉-alkynylcarbonyl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, a        protective group which is customary in nucleotide chemistry, or        a radical of the formula IIIa    -   R^(1a) is hydrogen, C₁-C₈-alkyl, C₂-C₁₈-alkenyl, C₂-C₁₈-alkynyl,        C₂-C₁₈-alkylcarbonyl, C₃-C₁₉-alkenylcarbonyl,        C₃-C₁₉-alkynylcarbonyl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, or a        radical of the formula IIIb    -   R² is hydrogen, hydroxyl, C₁-C₁₈-alkoxy, C₁-C₆-alkenyloxy, in        particular allyloxy, halogen, in particular F, azido or NH₂;    -   a is oxy, sulfanediyl or methylene;    -   n is an integer≧1;    -   W is oxo, thioxo or selenoxo;    -   V is oxy, sulfanediyl or imino;    -   Y is oxy, sulfanediyl, imino or methylene;    -   Y′ is oxy, sulfanediyl, imino, (CH₂)_(m) or V(CH₂)_(m), in which    -   m is an integer from 1 to 18;    -   X is hydroxyl or mercapto;    -   U is hydroxyl, mercapto, SeH, C₁-C₁₈-alkoxy, C₁-C₁₈-alkyl,        C₆-C₂₀-aryl, (C₆-C₁₄)aryl-(C₁-C₈)-alkyl, NHR³, NR³R⁴ or a        radical of the formula IV        (OCH₂CH₂)_(p)O(CH₂)_(q)CH₂R⁵  (IV)    -    in which    -   R³ is C₁-C₁₈-alkyl, C₆-C₂₀-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl,        —(CH₂)_(c)—[NH(CH₂)_(c)]_(d)—NR⁶R⁶, in which c is an integer        from 2 to 6 and d is an integer from 0 to 6, and R⁶ is,        independently of each other, hydrogen or C₁-C₆-alkyl or        C₁-C₄-alkoxy-C₁-C₆-alkyl;    -   R⁴ is C₁-C₁₈-alkyl, C₆-C₂₀-aryl or (C₆-C₁₀)-aryl-(C₁-C₈)-alkyl,        or, in the case of NR³R⁴, is, together with R³ and the nitrogen        atom carrying them, a 5-6-membered heterocyclic ring which can        additionally contain a further heteroatom from the series O, S        and N,    -   p is an integer from 1 to 100,    -   q is an integer from 0 to 22,    -   R⁵ is hydrogen or a functional group such as, for example,        hydroxyl, amino, C₁-C₁₈-alkylamino, COOH, CONH₂,        COO(C₁-C₄)-alkyl or halogen;    -   Z and Z′ are, independently of each other, hydroxyl, mercapto,        SeH, C₁-C₂₂-alkoxy, —O—(CH₂)_(b)—NR⁶R⁷, in which b is an integer        from 1 to 6, and R⁷ is C₁-C₆-alkyl or R⁶ and R⁷, together with        the nitrogen atom carrying them, form a 3-6-membered ring,        C₁-C₁₈-alkyl, C₆-C₂₀-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl,        (C₆-C₁₄)-aryl-(C₁-C₈)-alkoxy, where is also heteroaryl and aryl        is optionally substituted by 1, 2 or 3 identical or different        radicals from the group carboxyl, amino, nitro,        C₁-C₄-alkylamino, C₁-C₆-alkoxy, hydroxyl, halogen and cyano,        C₁-C₁₈-alkylmercapto, NHR³, NR³R⁴, a radical of the formula IV        or a group which favors intracellular uptake or serves for        labeling a DNA probe or, when the oligonucleotide analog        hybridizes to the target nucleic acid, attacks the latter while        binding, cross-linking or cleaving, and        the curved bracket indicates that R² and the adjacent phosphoryl        or —Y′—R^(1a) radical can either be located in the 2′ and 3′        positions or else, conversely, in the 3′ and 2′ position, with        it being possible for each nucleotide to be present in its D or        L configuration, and for the base B to be located in the α or β        position.

DETAILED DESCRIPTION

Bases which are customary in nucleotide chemistry are to be understoodto mean, for example, natural bases, such as adenine, cytosine, thymine,guanine, uracil or hypoxanthine, or unnatural bases, such as, forexample, purine, 8-azapurine, 2,6-diaminopurine, 7-deazaadenine,7-deazaguanine, N⁴,N⁴-ethanocytosine, N⁶,N⁶-ethano-2,6-diaminopurine,pseudoisocytosine, 5-methylcytosine, 5-fluorouracil,5-(C₃-C₆)-alkynyluracil, 5-(C₃-C₆)-alkynylcytosine, or their prodrugforms.

Oligonucleotides of the formula I are preferred which possess at leastone 7-deazaguanine base (E is NH₂ and F is OH) or 7-deazaadenine base (Eis H and F is NH₂) which is substituted at the 7 position.

Oligonucleotides of the formula I are particularly preferred whichpossess at least one 7-deazaadenine base which is substituted at the 7position and, where appropriate, one or more 7-deazaguanine bases whichare substituted at the 7 position, in addition.

Oligonucleotides of the formula I which possess at least one7-deazapurine base which is substituted at the 7 and 8 positions(=disubstituted 7-deazapurine bases) represent a further preferredembodiment of the present invention. Oligonucleotides of the formula Ihaving disubstituted 7-deazapurine bases are preferred in which thedisubstituted 7-deazapurine bases carry a substituent at the 8 positionwhich is defined under R¹⁶ 2., 3., 4., 5., 14. and 15. A halogen, forexample fluorine, is particularly preferred at the 8 position. Thesubstituents defined under R¹⁵ 3., 4., 5., 14. and 15., in particularhexynyl, are preferred substituents at the 7 position. Oligonucleotidesof the formula I are also preferred in which

-   -   R¹⁵ is        -   1. NO₂,        -   2. NH₂,        -   3. —S—(C₁-C₆)-alkyl,        -   4. (C₁-C₆)-alkoxy,        -   5. (C₆-C₂₀)-aryloxy,        -   6. SiH₃,        -   8. (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or (C₂-C₁₀)-alkynyl            which is substituted by one or more radicals from the group            SH, S—(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —NR(c)R(d),            —CO—R(b), —NH—CO—NR(c)R(d), —NR(c)R(g), —NR(e)R(f) or            —NR(e)R(g), or by a polyalkylene glycol radical of the            formula —[O—(CH₂)_(r)]_(s)—NR(c)R(d), where r and s are,            independently of each other, an integer between 1 and 18,            preferably 1 and 6, with it being possible for functional            groups such as OH, SH, —CO—R(b), —NH—CO—NR(c)R(d),            —NR(c)R(d), —NR(e)R(f), —NR(e)R(g) or —NR(c)R(g)            additionally to be linked to one or more groups, where            appropriate via a further linker, which favor intracellular            uptake or serve for labeling a DNA or RNA probe or, when the            oligonucleotide analog hybridizes to the target nucleic            acid, attack the latter while binding, cross-linking or            cleaving, or        -   9. (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or (C₂-C₁₀)-alkynyl in            which from one to all the H atoms are substituted by            halogen, preferably fluorine; and    -   R¹⁶ is hydrogen.

If only the 7 position of the 7-deazapurine bases is substituted(R¹⁶=H), C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl or C₂-C₁₀-alkynyl radicals, inwhich from one to all the H atoms are substituted by halogen, preferablyfluorine, are then particularly preferred for the 7 position.

Generally, 7-deazapurine-containing oligonucleotides are preferred inwhich the 7-deazapurine bases bear electron-attracting substituents atthe 7 position and/or 8 position.

Oligonucleotides of the formula I are also preferred in which the baseis located in the β position on the sugar, the nucleotides are in the Dconfiguration, R² is located in the 2′ position and a is oxy.

The 7 position of the azapurine ring system is to be understood to meanthe position at which the substituent R¹⁵ is located. Correspondingly,the substituent R¹⁶ is located at the 8 position.

When attaching to complementary nucleic acids (target nucleic acids),the novel oligonucleotides exhibit a binding affinity which is superiorto that exhibited by the natural oligonucleotides.

A further advantage of the novel oligonucleotides is that theirstability towards acids and nucleases is increased as compared with thatof oligonucleotides which contain natural purine bases.

It is advantageous if additional modifications, for example of thephosphate backbone, the ribose unit or the oligonucleotide ends, areintroduced into these oligonucleotides when they are to be usedtherapeutically [J. S. Cohen, Topics in Molecular and Structural Biology12 (1989) Macmillan Press, E. Uhlmann et al., above]. For example,modifications, which are known per se, of the sugar phosphate backboneresult in the novel oligonucleotides becoming even more efficientlyprotected against nuclease attack, which is advantageous.

Compounds of the formula I are also preferred, therefore, in which V, Y,Y′ and W have the meaning of thioxo, selenoxo, oxy, oxo, sulfanediyl,imino or methylene, and U has the meaning of hydroxyl, mercapto ormethyl. These compounds are very particularly preferred if R²additionally is hydroxyl or hydrogen, in particular hydrogen.

Compounds of the formula I in which R¹ and R^(1a) are hydrogen alsorepresent a preferred embodiment.

Compounds of the formula I are very particularly preferred in which R¹and/or R^(1a) is hydrogen, R² is hydroxyl or hydrogen, U is hydroxyl ormercapto, and V, Y, Y′ and W have the meaning of thioxo, oxy, oxo orhydroxyl.

Protective groups which are customary in nucleotide chemistry are to beunderstood to mean, for example, amino protective groups, hydroxylprotective groups or other protective groups as described in [E.Sonveaux, 1986, Bioorganic Chemistry, 14, 274-325 or S. L. Beaucage etal., 1992, Tetrahedron, 48, 2223-2311].

Alkyl, alkenyl and alkynyl may be straight-chain or branched. The samealso applies, in a corresponding manner, to radicals which are derivedfrom them, such as alkanoyl or alkoxy.

(C₁-C₁₀)-Alkyl is, in particular, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, pentyl, hexyl and heptyl. Examples of halogenated(C₁-C₁₀)-alkyls are CHF₂, CF₃, CH₂F, CF₃—CH₂—CH₂—, CF₃—CF₂—CH₂,CF₃(CF₂)₆—CH₂,

(C₂-C₁₀)-Alkenyl is, for example, vinyl (—CH═CH₂), 1-propenyl(—CH═CH—CH₃), 2-methyl-1-propenyl (—CH═C(CH₃)—CH₃), 1-butenyl(—CH═CH—CH₂—CH₃), 1-pentenyl, 1-hexenyl, 1-heptenyl and 1-octenyl.Examples of halogenated (C₂-C₁₀)-alkenyls are —CH═CF₂, —CH═CH—CF₃ and—CF═CF—CF₃.

(C₂-C₁₀)-Alkynyl is, for example, ethynyl (—C≡CH), 1-propynyl(—C≡C—CH₃), 1-butynyl (—C≡C—CH₂—CH₃), 3-methyl-butynyl(—C≡C—CH(CH₃)—CH₃), 3,3-dimethyl-butynyl (—C≡C—C(CH₃)₃), 1-pentynyl,1,3-pentadiynyl (—C≡C—C≡C—CH₃), 1-hexynyl and 1-heptynyl. Examples ofhalogenated (C₂-C₁₀)-alkynyls are —C≡C—CH₂F, —C≡C—CF₃, —C≡C—(CH₂)₃—CF₃and —C≡C—(CF₂)₃—CF₃.

Cycloalkyl is also understood to mean alkyl-substituted rings.

(C₆-C₂₀)-Aryl is, for example, phenyl, naphthyl or biphenylyl,preferably phenyl.

Halogen is to be understood to mean iodine, bromine, chlorine orfluorine.

Heteroaryl is understood to mean, in particular, radicals which arederived from phenyl or naphthyl in which one or more CH groups arereplaced by N and/or in which at least two adjacent CH groups arereplaced (with the formation of a five-membered aromatic ring) by S, NHor O. In addition, one or both atoms of the condensation site ofbicyclic radicals can be N atoms (as in indolizinyl).

Heteroaryl is, in particular, furanyl, thienyl, pyrrolyl, imidazolyl,pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl,indazolyl, quinolyl, isoquinolyl, phthalazinyl, quinoxalinyl,quinazolinyl and cinnolinyl.

The morpholinyl radical and the imidazolidinyl radical may be mentionedas examples of NR³R⁴ groups in which R³ and R⁴, together with thenitrogen atom carrying them, form a 5- to 6-membered heterocyclic ringwhich additionally contains a further heteroatom.

Physiologically tolerated salts of compounds of the formula (I) areunderstood to mean both inorganic and organic salts, as described inRemington's Pharmaceutical Sciences (17th edition, page 1418 (1985)).

Owing to their physical and chemical stability, and their solubility,sodium salts, potassium salts, calcium salts and ammonium salts, interalia, are preferred for acidic groups.

The invention is not limited to α- and β-D- or L-ribofuranosides, α- andβ-D- or L-deoxyribofuranosides and corresponding carbocyclic five-ringanalogs, but also applies to oligonucleotide analogs which are assembledfrom other sugar building blocks, for example xylofuranose andarabinofuranose derivatives, ring-expanded and ring-contracted sugars,and acyclic and ring-bridged sugar derivatives or suitable sugarderivatives of a different kind. Furthermore, the invention is notlimited to the derivatives of the phosphate radical which are listed byway of example in formula I, but also relates to the known dephosphoderivatives.

Consequently, the novel oligonucleotides can result from modifying thenatural structure in a variety of ways. Examples of such modifications,which are introduced by methods which are known per se, are:

a) Modifications of the Phosphate Bridge

The following may be mentioned by way of example: phosphorothioates,phosphorodithioates, methylphosphonates, phosphoroamidates,boranophosphates, methyl phosphates, ethyl phosphates andphenylphosphonates. Phosphorothioates, phosphorodithioates andmethylphosphonates are preferred modifications of the phosphate bridge.

b) Replacement of the Phosphate Bridge

The following may be mentioned by way of example: replacement withacetamide, formacetal, 3′-thioformacetal, methylhydroxylamine, oxime,methylenedimethylhydrazo, dimethylenesulfone and silyl groups.Replacement with acetamide, formacetals and 3′-thioformacetals ispreferred.

c) Modifications of the Sugar

The following may be mentioned by way of example: α-anomeric sugars,2′-O-methylribose, 2′-O-butylribose, 2′-O-allylribose,2′-fluoro-2′-deoxyribose, 2′-amino-2′-deoxyribose, α-arabinofuranose andcarbocyclic sugar analogs. The preferred modification is that due to2′-O-methylribose and 2′-O-n-butylribose.

d) Modifications of the Sugar and of the Phosphate Bridge

Those which may be mentioned by way of example are the peptide nucleicacids (PNA's), in which the sugar/phosphate backbone is replaced by anaminoethylglycine backbone (see German Patent Application P4408531.1),and the carbamate-bridged morpholino oligomers. The PNA's can also belinked to nucleic acids, as described in German Patent ApplicationP4408528.1.

e) Other Modifications of the Bases, in Particular of the PyrimidineBases

The following may be mentioned by way of example:

-   5-propynyl-2′-deoxyuridine, 5-propynyl-2′-deoxycytidine,    5-hexynyl-2′-deoxyuridine, 5-hexynyl-2′-deoxycytidine,    5-fluoro-2′-deoxycytidine, 5-fluoro-2′-deoxyuridine,    5-hydroxymethyl-2′-deoxyuridine, 5-methyl-2′-deoxycytidine and    5-bromo-2′-deoxycytidine. 5-Propynyl-2′-deoxyuridine,    5-hexynyl-2′-deoxyuridine, 5-hexynyl-2′-deoxycytidine and    5-propynyl-2′-deoxycytidine are preferred modifications.    f) 3′-3′ and 5′-5′ Inversions [e.g. M. Koga et al., J. Org. Chem.    56 (1991) 3757]    g) 5′ conjugates and 3′-conjugates.

Examples of groups which favor intracellular uptake are differentlipophilic radicals, such as —O—(CH₂)_(x)—CH₃, in which x is an integerfrom 6 to 18, —O—(CH₂)_(n)—CH═CH—(CH₂)_(m)—CH₃, in which n and m are,independently of each other, an integer from 6 to 12,—O—(CH₂CH₂O)₄—(CH₂)₉—CH₃, —O—(CH₂CH₂O)₈—(CH₂)₁₃—CH₃ and—O—(CH₂CH₂O)₇—(CH₂)₁₅—CH₃, and also steroid radicals, such ascholesteryl, or vitamin radicals, such as vitamin E, vitamin A orvitamin D, and other conjugates which exploit natural carrier systems,such as bile acid, folic acid, 2-(N-alkyl, N-alkoxy)-aminoanthraquinoneand conjugates of mannose and peptides of the corresponding receptorswhich lead to receptor-mediated endocytosis of the oligonucleotides,such as EGF (epidermal growth factor), bradykinin and PDGF (plateletderived growth factor). Labeling groups are to be understood to meanfluorescent groups, for example of dansyl(=N-dimethyl-1-aminonaphthyl-5-sulfonyl) derivatives, fluoresceinderivatives or coumarin derivatives, or chemiluminescent groups, forexample of acridine derivatives, and also the digoxygenin system, whichis detectable by means of ELISA, the biotin group, which is detectableby means of the biotin/avidin system, or else linker arms havingfunctional groups which permit subsequent derivatization with detectablereporter groups, for example an aminoalkyl linker which is convertedinto the chemiluminescence probe using an acridinium active ester. Othersuitable linkers are known to a person skilled in the art from thepublished patent applications EP 251786 and WO 93/09217.

h) Conjugation by Way of the 7 Position and/or the 8 Position on the7-Deazapurine

Groups which serve to label a DNA or RNA probe or which favorintracellular uptake can also be conjugated by way of the 7 positionand/or 8 position of the 7-deazapurine. 7-Deazapurine nucleosides towhich biotin or iminobiotin radicals are conjugated by way of the 7position of the 7-deazapurine, via a special connecting group, have beendisclosed by EP 63 879.

Labeling groups for a DNA or RNA probe are to be understood to meanfluorescent groups, for example of dansyl(=N-dimethyl-1-aminonaphthyl-5-sulfonyl) derivatives, fluoresceinderivatives or coumarin derivatives, or chemiluminescent groups, forexample of acridine derivatives, and also the digoxygenin system, whichis detectable by means of ELISA, or the biotin group, which isdetectable by means of the biotin/avidin system, and also theintercalators and chemically active groups which have already beenlisted under g) (see, also, Beaucage et al., Tetrah. (1993) Vol. 49, No.10, 1925-1963).

Examples of groups which favor intracellular uptake are steroidradicals, such as cholesteryl, or vitamin radicals such as vitamin E,vitamin A or vitamin D, and other conjugates which exploit naturalcarrier systems, such as bile acid, folic acid, 2-(N-alkyl,N-alkoxy)-aminoanthraquinone and conjugates of mannose and peptides ofthe corresponding receptors which lead to receptor-mediated endocytosisof the oligonucleotides, such as EGF (epidermal growth factor),bradykinin and PDGF (platelet derived growth factor).

In a general manner, the described groups can be introduced either atthe level of the oligonucleotides (for example by way of SH groups) orat the level of the monomers (phosphonates, phosphoamidites ortriphosphates). In the case of the monomers, in particular in the caseof the triphosphates, it is advantageous to leave the Bide chains, intowhich a reporter group or an intercalator group is to be introduced, inthe protected state, and only to eliminate the side-chain protectivegroups, and to react with an optionally activated derivative of thecorresponding reporter group or intercalator group, after thephosphorylation.

Typical labeling groups are:

Oligonucleotide analogs which bind to nucleic acids or intercalate withthem and/or cleave or cross-link them, contain, for example, acridine,psoralene, phenanthridine, naphthoquinone, daunomycin orchloroethylaminoaryl conjugates. Typical intercalating and cross-linkingradicals are:

The invention furthermore relates to compounds of the formula V

in which

-   -   V is oxy, sulfanediyl or imino;    -   Y^(b) is oxy, sulfanediyl, imino or methylene;    -   a is oxy, sulfanediyl or methylene;    -   R^(2b) is hydrogen, OR¹², C₁-C₁₈-alkoxy, C₁-C₆-alkenyloxy, in        particular allyloxy, halogen, azido or NR¹⁰R¹¹;    -   R¹ is a protective group which is customary in nucleotide        chemistry;    -   R^(1b) is a succinyl radical or other conventional linker for        linking the oligonucleotide containing this group to a solid        support e.g., an amino-functionalized or        methylamino-functionalized support, by way of an amide or        methylimide bond, or the like, or    -   is a radical of the formula IIIc or IIId        in which    -   U is (C₁-C₁₈)-alkoxy, (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl,        (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, O—R⁷, S—R⁷ or a radical of the        formula IV        (OCH₂CH₂)_(p)O(CH₂)_(q)CH₂R⁵  (IV)    -    in which R⁵ is H;    -   Q is a radical —NR⁸R⁹,    -   R⁷ is —(CH₂)₂—CN;    -   R⁸ and R⁹ are identical or different and are C₁-C₆-alkyl, in        particular isopropyl or ethyl, or, together with the nitrogen        atom carrying them, are a 5-9-membered heterocyclic ring which        can additionally contain a further hetero atom from the series        O, S and N, in particular    -   E′ and F′ are, independently of each other, H, OR¹² or NR¹⁰R¹¹,    -   R¹⁰ and R¹¹ are identical or different and are hydrogen or an        amino protective group which is customary in nucleotide        chemistry, or R¹⁰ and R¹¹ together form an amino protective        group which is customary in nucleotide chemistry,    -   R¹² is hydrogen or a hydroxyl protective group which is        customary in nucleotide chemistry, such as, for example,        t-butyldimethyl-silyl, dimethoxytriphenyl-methyl (DMT),        triisopropyl-silyl, o-nitro-benzyl, p-nitro-benzyl, iBu,        2-fluorophenyl-4-methoxy-piperidin-4-yl (FPMP), or methyl,    -   R¹⁵ and R¹⁶ are, independently of each other,        -   1. hydrogen,        -   2. halogen,        -   3. (C₁-C₁₀)-alkyl,        -   4. (C₂-C₁₀)-alkenyl,        -   5. (C₂-C₁₀)-alkynyl,        -   6. NO₂,        -   7. NH₂,        -   8. cyano,        -   9. —S—(C₁—C₆)-alkyl,        -   10. (C₁-C₆)-alkoxy,        -   11. (C₆-C₂₀)-aryloxy,        -   12. SiH₃,        -   14. a radical as defined under 3., 4. or 5. which is            substituted by one or more radicals from the group SH,            S—(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —NR(c)R(d), —CO—R(b),            —NH—CO—NR(c)R(d), —NR(c)R(g), —NR(e)R(f) or —NR(e)R(g), or            by a polyalkyleneglycol radical of the formula            —[O—(CH₂)_(r)]_(s)—NR(c)R(d), where r and s are,            independently of each other, an integer between 1 and 18,            preferably 1 and 6, with it being possible for functional            groups such as OH, SH, —CO—R(b), —NH—CO—NR(c)R(d),            —NR(c)R(d), —NR(e)R(f), —NR(e)R(g) or —NR(c)R(g) to carry a            protective group which is customary in nucleotide chemistry            or to be linked, where appropriate via a further linker, to            one or more groups which favor intracellular uptake or serve            as labeling for a DNA or RNA probe or, when the            oligonucleotide analog hybridizes to the target nucleic            acid, attack the latter while binding, cross-linking or            cleaving, or        -   15. a radical as defined under 3., 4. or 5. in which from            one to all the H atoms are substituted by halogen,            preferably fluorine.        -   R(a) is OH, (C₁-C₆)-alkoxy, (C₆-C₂₀)-aryloxy, NH₂ or NH-T,            where T is an alkylcarboxyl group or alkylamino group which            is linked, optionally via a further linker, to one or more            groups which favor intracellular uptake, or serve as            labeling for a DNA or RNA probe or, when the oligonucleotide            analog hybridizes to the target nucleic acid, attack the            latter while binding, cross-linking or cleaving,        -   R(b) is hydroxyl, (C₁-C₆)-alkoxy or —NR(c)R(d),        -   R(c) and R(d) are, independently of each other, H or            (C₁-C₆)-alkyl which is unsubstituted or substituted by            —NR(e)R(f) or —NR(e)R(g),        -   R(e) and R(f) are, independently of, each other, H or            (C₁-C₆)-alkyl,        -   R(g) is (C₁-C₆)-alkyl-COOH,        -   with the proviso that R¹⁵ and R¹⁶ cannot each simultaneously            be hydrogen, NO₂, NH₂, cyano or SiH₃,            with functional groups such as OH, NH₂ or COOH being            protected, where appropriate, with a protective group which            is customary in nucleotide chemistry, and the curved bracket            indicating that R^(2b) and the adjacent —Y^(b)—R^(1b)            radical can be located in the 2′ and 3′ positions or else,            conversely, in the 3′ and 2′ positions.

A preferred embodiment is represented by compounds of the formula (V) inwhich V, Y^(b) and a are oxy, R^(2b) is hydrogen or OR¹², in particularhydrogen, and R^(1b) is a radical of the formula (IIIc) or (IIId), withU being an O—(CH₂)₂—CN, and R⁸ and R⁹ being identical or, different andbeing isopropyl or ethyl, or, together with the N atom carrying them,being an aliphatic heterocycle, preferably pyrrolidino. These compoundsare very particularly preferred if, in addition, the base is located inthe β position on the sugar and R^(2b) is located in the 2′ position.

Compounds of formula (V) are also preferred in which E is NR¹⁰R¹¹ and Fis H, and, quite generally, those compounds of the formula (V) arepreferred which can be employed for preparing preferred oligonucleotidesof the formula I.

Examples of preferred amino protective groups are acyl or amidineprotective groups.

The radical of the formula (IIId) which is customarily present as a saltis to be understood to mean inorganic or organic salts, for examplealkali metal, alkaline earth metal or ammonium salts, which aredescribed, for example, in Remington's Pharmaceutical Sciences (17thedition, page 1418 (1985)). Triethylammonium and pyridinium salts may bementioned by way of example. However, the invention also embracescompounds of the formula (V) in which the radical of the formula (IIId)is present as a free acid.

The compounds of the formula V may be employed as structural componentsfor preparing the novel oligonucleotides of the formula I.

EP 251 786 discloses 7-deazapurine nucleotides, and theirmonophosphates, diphosphates or triphosphates, which possess analkynylamino group at the 7-purine position. The alkynylamino groupserves as a linker by way of which fluorescent labeling molecules can becoupled to the nucleotide. The dideoxynucleotides which have beenprovided with a fluorescence label can then be used as chain terminatormolecules for dideoxy sequencing in accordance with Sanger and detecteddirectly by means of fluorescence spectroscopy. U.S. Pat. No. 5,241,060discloses 7-deazapurine nucleotides which carry a detectable radical onthe 7-deazapurine.

The invention also relates to compounds of the formula VI

in which, independently of each other,

-   -   U′=U″=U′″ is hydroxyl or mercapto, and U′ can additionally be        BH₃,    -   e and f are 0 or 1;    -   R¹³ is hydrogen, OH, C₁-C₁₈-alkoxy, or C₁-C₁₆-alkenyloxy, in        particular allyloxy;    -   E and F are, independently of each other, H, OH or NH₂; and    -   R¹⁵ and R¹⁶ are, independently of each other,        -   1. hydrogen,        -   2. halogen,        -   3. (C₁-C₁₀)-alkyl,        -   4. (C₂-C₁₀)-alkenyl,        -   5. (C₂-C₁₀)-alkynyl,        -   6. NO_(2,)        -   7. NH₂,        -   8. cyano,        -   9. —S—(C₁-C₆)-alkyl,        -   10. (C₁-C₆)-alkoxy,        -   11. (C₆-C₂₀)-aryloxy,        -   12. SiH₃,        -   14. a radical as defined under 3., 4. or 5. which is            substituted by one or more radicals from the group SH,            S—(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —NR(c)R(d), —CO—R(b),            —NH—CO—NR(c)R(d), —NR(c)R(g), —NR(e)R(f) or —NR(e)R(g), or            by a polyalkyleneglycol radical of the formula            —[O—(CH₂)_(r)]_(s)—NR(c)R(d), where r and s are,            independently of each other, an integer between 1 and 18,            preferably 1 and 6, with it being possible for functional            groups such as OH, SH, —CO—R(b), —NH—CO—NR(c)R(d),            —NR(c)R(d), —NR(e)R(f), —NR(e)R(g) or —NR(c)R(g)            additionally to be linked, where appropriate via a further            linker, to one or more groups which favor intracellular            uptake or serve as labeling for a DNA or RNA probe or, when            the oligonucleotide analog hybridizes to the target nucleic            acid, attack the latter while binding, cross-linking or            cleaving, or        -   15. a radical as defined under 3., 4. or 5. in which from            one to all the H atoms are substituted by halogen,            preferably fluorine.        -   R(a) is OH, (C₁-C₆)-alkoxy, (C₆-C₂₀)-aryloxy, NH₂ or NH-T,            with T representing an alkylcarboxyl or alkylamino group            which is linked, where appropriate via a further linker, to            one or more groups which favor intracellular uptake or serve            as labeling for a DNA or RNA probe or, when the            oligonucleotide analog hybridizes to the target nucleic            acid, attack the latter while binding, cross-linking or            cleaving,        -   R(b) is hydroxyl, (C₁-C₆)-alkoxy or —NR(c)R(d),        -   R(c) and R(d) are, independently of each other, H or            (C₁-C₆)-alkyl which is unsubstituted or substituted by            —NR(e)R(f) or —NR(e)R(g),        -   R(e) and R(f) are, independently of each other, H or            (C₁-C₆)-alkyl,        -   R(g) is (C₁-C₆)-alkyl-COOH,        -   with the proviso that R¹⁵ and R¹⁶ cannot each simultaneously            be hydrogen, NO₂, NH₂, cyano or SiH₃,            with compounds of the formula VI being excepted in which R¹⁶            is H and R¹⁵ is (C₂-C₁₀)-alkynyl which is substituted by            —NR(c)R(d) or —NR(e)R(f); and with the additional proviso            that e and f are not 0 if E is OH or NH₂ and F is OH, R¹⁶ is            hydrogen and R¹⁵ is Br, Cl, F, cyano, (C₁-C₄)-alkyl,            (C₂-C₄)-alkenyl or (C₂-C₄)-alkynyl.

The invention also embraces compounds of the formula VI which areprovided, in a generally customary manner, with a radioactive label (forexample, αP atom is ³²P; U′ is ³⁵S).

Compounds of the formula VI are preferred in which U′ is hydroxyl ormercapto, U″=U′″. is hydroxyl, and e and/or f is 1. Compounds of theformula VI are particularly preferred when e and f are 1.

The compounds of the formula VI which are customarily present as a saltcomprise inorganic or organic salts, for example alkali metal, alkalineearth metal or ammonium salts [Remington's Pharmaceutical Sciences (17thedition, page 1418 (1985)]. Triethylammonium and pyridinium salts may bementioned by way of example. The novel VI compounds also comprise thosecompounds in which the phosphate group is present as a free acid.

The novel compounds of the formula VI may be employed generally as aidsin molecular biology, for example in PCR reactions (e=f=1, R¹³=OH) orfor sequencing (e=f=1; R¹³=H or OH). In PCR-reactions compounds of theformula VI are preferred in which R¹⁶ is H and R¹⁵ is halogen.Amplification of longer nucleodide sequences is enhanced using themodified oligonucleotides.

The use of the novel 7-deazapurine nucleotides for sequencing nucleicacids is advantageous for several reasons. Thus, the band compressionwhich can often be observed in GC-rich nucleotide regions in the Sangersequencing method (dideoxy technique), and which hinders correctdetermination of the nucleotide sequence, is either eliminated or atleast reduced. In addition, the double-stranded nucleic acids which aresynthesized by DNA polymerases or RNA polymerases during the sequencingare stabilized by the incorporation of 7-, 8- or 7,8-substituted7-deazapurine bases. It is consequently more advantageous to usesubstituted 7-deazapurine nucleotides than to use unsubstituted7-deazaguanosine nucleotides, which are customarily employed in nucleicacid sequencing in order to eliminate band compressions in GC-rich DNAstretches (EP 212536). A further advantage of using substituted7-deazapurine nucleotides in the sequencing is that fluorescent residuesin the form of reporter groups, which make possiblefluorescence-spectroscopic detection of the nucleic acid molecules whichare synthesized during the sequencing reaction, can be introduced ontothe substituents in a series of subsequent reactions.

In addition, the incorporation of self-fluorescent, substituted7-deazapurine bases into oligonucleotides renders it possible to detectthe latter directly by way of the self-fluorescence of the substituted7-deazapurine bases. Thus, the 7-deazapurine bases, which inunsubstituted form are not fluorescent, become fluorescent, for example,when an alkynyl group, for example hexynyl, is introduced at the 7position. The self-fluorescence of these compounds can be measured at350 nm (emission) following excitation with light of 280 nm wavelength.

The compounds of the formula VI can be prepared by proceeding from thecorresponding substituted 7-deazapurine nucleosides and using well knownmethods. The compounds of the formula VI can preferably be prepared byan abbreviated one-pot method due to Ludwig, in the presence of1,8-bis(dimethylamino)naphthalene and trimethyl phosphate [J. Ludwig etal., (1981) Acta Biochem. Biophys. Sci. Hung., 16, 131].

The invention also relates to compounds of the formula VII

in which

-   -   E and F are, independently of each other, H, OH or NH₂, and OH        and NH₂ are, where appropriate, protected by a protective group        which is customary in nucleotide chemistry;    -   R¹⁵ and R¹⁶ are, independently of each other, hydrogen,        (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl, (C₂-C₁₀)-alkynyl, I, Cl, Br,        F, cyano, or (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or        (C₂-C₁₀)-alkynyl in which from one to all the H atoms are        substituted by halogen, preferably fluorine, with it not being        possible for R¹⁵ and R¹⁶ to be simultaneously hydrogen and        cyano, and with the further proviso that R¹⁵ is not I if R¹⁶ is        hydrogen, E is NH₂ and F is OH,    -   R¹⁴ are, independently of each other, H or a protective group        which is customary in nucleotide chemistry.

The invention also embraces all the tautomeric forms of the compounds ofthe formulae I, V, VI and VII, and, in particular, all the tautomericforms of the 7-deazapurine bases of the formula II.

In a quite general manner, those compounds of the formulae V, VI and VIIare also preferred which can be used as starting compounds orintermediates for the preparation of preferred oligonucleotides of theformula I.

The invention furthermore relates to a process for preparing the noveloligonucleotides of the formula I. The standard conditions which arecustomary in the chemical synthesis of oligonucleotides can be appliedfor preparing the novel oligonucleotides containing substituted7-deazapurine.

The novel oligonucleotides of the formula I are prepared in solution or,preferably, on a solid phase, where appropriate using an automaticsynthesis device. The oligomers of the formula I can be assembledstepwise by successively condensing a mononucleotide, which in each casepossesses a nucleotide base, onto an appropriately derivatized supportor onto a growing oligomer chain. Alternatively, the oligonucleotides ofthe formula I can be assembled by joining dinucleotides ortrinucleotides together [S. Beaucage et al., Tetrah. vol. 48, No. 12,2223-2311, (1992); and Tetrah. vol. 48, No. 28, 6123-6194, (1993)]. Thisis particularly advantageous when synthesizing oligonucleotides whichpossess modified phosphate bridges.

The oligonucleotides are assembled using methods which are known to theperson skilled in the art, such as the triester method, theH-phosphonate method or the phosphoramidite method [E. Sonveaux, (1986),Bioorganic Chemistry, 14, 274-325; S. L. Beaucage et al., (1992),Tetrahedron, 48, 2223-2311]. The nucleotide monomer structuralcomponents of the formula V, particularly preferably those of theformula V in which E′ is NR¹⁰R¹¹ and F′ is OR¹², or F′ is NR¹⁰NR¹¹ andE′ is H, are preferably employed for introducing the 7-deazapurinederivatives.

The compounds of the formula V can be prepared, as structural componentsfor the oligonucleotide solid phase synthesis, by proceeding from thecorresponding 7-deazapurine nucleosides. Substituents can be introducedat the 7 position of the 7-deazapurine ring system using well-knownmethods. For example, the preparation of 7-deazapurine nucleosides whichare substituted at the 7 position by halogen or methyl is described bySeela et al. [Helvetica Chimica Acta, (1994) 77, 897-903]. Alkenyl- oralkynyl-substituted 7-deazapurine derivatives of: the formula V can beprepared by proceeding from the known 5-iodotubercidin(=7-I-7-deazaadenosine, see Seela et al., above), and coupling alkenylor alkynyl groups onto the 7 position of the 7-deazapurine ring systemby means of a cross-coupling reaction in the presence oftetrakis(triphenylphosphine)palladium(O). Electrophilic substituents(for example halogens) can be introduced into the 8 position of the7-deazapurine ring system if nucleosides are employed as startingcompounds which possess an electron-supplying substituent (for examplean amino group) at the 2 position of the 7-deazapurine. If the 2-aminogroup is, for example, acetylated, the electrophilic substituent is thendirected into the 7 position. Consequently, the present invention alsorelates to a process for the regioselective insertion of electrophilicsubstituents (for example halogens) into the 7 or 8 position of7-deazanucleosides. The halogenated nucleosides can then be used asstarting compounds for the insertion of other substituents, for examplealkyl, alkenyl or alkynyl groups, by means of the above-describedpalladium-catalyzed cross-coupling reaction. Alkoxy derivatives orsubstituted amine derivatives can be introduced by nucleophilicsubstitution, and nitro groups can be introduced by electrophilicsubstitution.

After suitable protective groups for the amino groups of the7-deazapurine bases and for the free 5′-hydroxyl group of the sugar havebeen introduced, the monomers are converted into the correspondingphosphonate or phosphoramidite derivatives. Suitable amino protectivegroups, for example in the form of a formamidine protective group((dimethylamino)methylidene) or acyl protective groups (e.g. benzoyl orphenoxyacetyl), are inserted using well-known methods [L. J. McBride etal., (1983) Tetrahedron Lett., 24, 2953, G. S. Ti et al., (1982) J. Am.Chem. Soc., 104, 1316; H. Schaller et al. (1963), J. Am. Chem. Soc., 85,3821], with it being advantageous, when the amino group is acylated, touse the Schaller peracylation method. An example of a suitableprotective group for the free 5′-OH group of the sugar is4,4′-dimethoxytrityl, whose insertion is likewise effected using knownmethods [C. B. Reese (1978), Tetrahedron, 34, 3143; D. Flockerzi et al.,(1981), Liebigs Ann. Chem., 1568]. The monomers which have beenprotected in this way can be converted into the correspondingphosphonates in accordance with a protocol due to Froehler et al. [B. C.Froehler et al., (1986), Nucl. Acid Res., 14, 5399].Cyanoethyl-phosphoramidite derivatives can, for example, be prepared byreacting the monomers withchloro-β-cyanoethoxy-(N,N-diisopropylamino)phosphane in anhydrousdichloromethane [N.D. Sinha et al., (1984) Nucl. Acid Res., 12, 4539].

Compounds of the formula I whose oligonucleotide moiety is modified atthe 3′ end and/or the 5′ end are synthesized, as regards thesemodifications, using the methods described in EP-A 0 552 766.

For use according to the invention, the oligonucleotides have a lengthof from 4 to 100, preferably of about 5-40, in particular of about 6-30,nucleotides. Otherwise, the above-described preference ranges,modifications and conjugations also apply in this case too.

The present invention relates to the use of oligonucleotides containingat least one substituted 7-deazapurine, preferably 7-deazaadenine or7-deazaguanine, as a diagnostic reagent, for example for detecting thepresence or absence of, or the quantity of, a specific double-strandedor single-stranded nucleic acid molecule in a biological sample. One ormore of these oligonucleotides maybe directly or indirectly bound orabsorbed onto a solid support, or provided as a solution in a solvent ordiluent, optionally together with other conventional diagnosticallyrelevant auxiliary reagents.

The invention furthermore relates to pharmaceutical compositionscomprising one or more oligonucleotides of the formula I, together witha physiologically acceptable excipient and, where appropriate, suitableadditives and/or conventional auxiliary substances.

In a quite general manner, the present invention extends to the use ofoligonucleotides of the formula —I in therapeutically effective amountsin improved therapeutic methods. In general, therapeutically effectiveoligonucleotide derivatives are understood to mean antisenseoligonucleotides, triple helix-forming oligonucleotides, aptamers orribozymes, in particular antisense oligonucleotides.

The pharmaceuticals of the present invention can, for example, be usedto treat diseases which are caused by viruses, for example by HIV,HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses.

Novel antisense oligonucleotide derivatives, that is antisenseoligonucleotides in which at least one purine base is replaced by asubstituted 7-deazapurine base, and which are effective against thesetargets, have, for example, the following base sequences:

a) Against HIV, e.g.

(SEQ ID NO.: 1) 5′-A C A C C C A A T T C T G A A A A T G G-3′ or (SEQ IDNO.: 2) 5′-A G G T C C C T G T T C G G G C G C C A-3′  or (SEQ ID NO.:3) 5′-G T C G A C A C C C A A T T C T G A A A A T G G A T A A-3′ or (SEQID NO.: 4) 5′-G C T A T G T C G A C A C C C A A T T C T G A A A-3′ or(SEQ ID NO.: 5) 5′-T C G T C G C T G T C T C C G C T T C T T C T T C C TG C C A-3′ or

The pharmaceuticals of the present invention are also suitable, forexample, for treating cancer. For example, oligonucleotide sequences canbe used in this context which are directed against targets which areresponsible for the occurrence of cancer or for cancer growth. Examplesof such targets are:

-   1) nuclear oncoproteins such as, for example, c-myc, N-myc, c-myb,    c-fos, c-fos/jun, PCNA and p120,-   2) cytoplasmic/membrane-associated oncoproteins such as, for    example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos,    c-src and c-abl,-   3) cellular receptors, such as, for example, the EGF receptor,    c-erbA, retinoid receptors, the protein kinase regulatory subunit    and c-fms,-   4) cytokines, growth factors, and extracellular matrix, such as, for    example, CSF-1, IL-6, IL-1a, IL-1b, IL-2, IL-4, bFGF, myeloblastin    and fibronectin.

Novel antisense oligonucleotides of the formula I which are effectiveagainst these targets have, for example, the following base sequences:

a) Against c-Ha-ras, e.g.

-   5′-CAGCTGCAACCCAGC-3′ (SEQ ID NO.:7)    c) c-myc, e.g.

(SEQ ID NO.: 8) 5′-G G C T G C T G G A G C G G G G C A C A C-3′ (SEQ IDNO.: 9) 5′-A A C G T T G A G G G G C A T-3′d) c-myb, e.g.

-   5′-GTGCCGGGGTCTTCGGGC-3′ (SEQ ID NO.: 10)    e) c-fos, e.g.

(SEQ ID NO.: 11) 5′-G G A G A A C A T C A T G G T C G A A A G-3′ (SEQ IDNO.: 12) 5′-C C C G A G A A C A T C A T G G T C G A A G-3′ (SEQ ID NO.:13 5′-G G G G A A A G C C C G G C A A C G G G-3′f) p120, e.g.

-   5′-CACCCGCCTTGGCCTCCCAC-3′ (SEQ ID NO.: 14)    g) EGF Receptor, e.g.

(SEQ ID NO.: 15) 5′-G G G A C T C C G G C G C A G C G C-3′ (SEQ ID NO.:16) 5′-G G C A A A C T T T C T T T T C C T C C-3′h) p53 Tumor Suppressor, e.g.

(SEQ ID NO.: 17) 5′-G G G A A G G A G G A G G A T G A G G-3′ (SEQ IDNO.: 18) 5′-G G C A G T C A T C C A G C T T C G G A G-3′

The pharmaceuticals of the present invention are furthermore suitable,for example, for treating diseases which are affected by integrins orcell-cell adhesion receptors, for example by VLA-4, VLA-2, ICAM, VCAM orELAM.

Novel antisense oligonucleotide derivatives which are effective againstthese targets have, for example, the following base sequences:

a) VLA-4, e.g.

-   5′-GCAGTAAGCATCCATATC-3′ (SEQ ID NO.: 19)    b) ICAM, e.g.

(SEQ ID NO.: 20) 5′-C C C C C A C C A C T T C C C C T C T C-3′ (SEQ IDNO.: 21) 5′-C T C C C C C A C C A C T T C C C C T C-3′ (SEQ ID NO.: 22)5′-G C T G G G A G C C A T A G C G A G G-3′c) ELAM-1, e.g.

(SEQ ID NO.: 23) 5′-A C T G C T G C C T C T T G T C T C A G G-3′ (SEQ IDNO.: 24) 5′-C A A T C A A T G A C T T C A A G A G T T C-3′

The pharmaceuticals of the present invention are also suitable, forexample, for preventing restenosis. For example, oligonucleotidesequences can be used in this context which are directed against targetswhich are responsible for proliferation or migration. Examples of thesetargets are:

-   1) Nuclear transactivator proteins and cyclins, such as, for    example, c-myc, c-myb, c-fos, c-fos/jun, cyclins and cdc2 kinase-   2) Mitogens or growth factors, such as, for example, PDGF, bFGF,    EGF, HB-EGF and TGF-β.-   3) Cellular receptors such as, for example, bFGF receptor, EGF    receptor and PDGF receptor.

Novel oligonucleotides of the formula I which are effective againstthese targets have, for example, the following base sequences:

a) c-myb

-   5′-GTGTCGGGGTCTCCGGGC-3′ (SEQ ID NO.: 25)    b) c-myc-   5′-CACGTTGAGGGGCAT-3′ (SEQ ID NO.: 26)    c) cdc2 kinase-   5′-GTCTTCCATAGTTACTCA-3′ (SEQ ID NO.: 27)    d) PCNA (proliferating cell nuclear antigen of rat)-   5′-GATCAGGCGTGCCTCAAA-3′ (SEQ ID NO.:28)

The pharmaceuticals can be used, for example, in the form ofpharmaceutical preparations which can be administered orally, forexample in the form of tablets, coated tablets, hard or soft gelatincapsules, solutions, emulsions or suspensions. The inclusion of thepharmaceuticals in liposomes, which, where appropriate, containadditional components such as proteins, likewise represents a suitableadministration form. They can also be administered rectally, for examplein the form of suppositories, or parenterally, for example in the formof injection solutions. For the production of pharmaceuticalpreparations, these compounds can be processed in therapeutically inert,organic and inorganic excipients. Examples of such excipients fortablets, coated tablets and hard gelatin capsules are lactose, cornstarch, or derivatives thereof, tallow and stearic acid, or saltsthereof. Suitable excipients for preparing solutions are water, polyols,sucrose, invert sugar and glucose. Suitable excipients for injectionsolutions are water, alcohols, polyols, glycerol and vegetable oils.Suitable excipients for suppositories are vegetable and hardened oils,waxes, fats and semiliquid polyols. The pharmaceutical preparations canalso contain preservatives, solvents, stabilizers, wetting agents,emulsifiers, sweeteners, colorants, flavorants, salts for altering theosmotic pressure, buffers, coating agents, antioxidants and othertherapeutical active compounds, where appropriate.

Preferred forms of administration are topical administrations, localadministrations, such as, for example, using a catheter, or elseinjections. For injection, the antisense oligonucleotide derivatives areformulated in a liquid solution, preferably in a physiologicallyacceptable buffer, such as, e.g., Hank's solution or Ringer's solution.However, the antisense oligonucleotides can also be formulated in solidform and dissolved or suspended prior to use. The doses which arepreferred for systemic administration amount to from about 0.01 mg/kg toabout 50 mg/kg of body weight and per day.

In a quite general manner, the invention extends to the use of compoundsof the formula I as DNA probes or primers in DNA diagnostics and, in ageneral manner, as aids in molecular biology , as noted earlier.

Individual DNA molecules can be visualized electron microscopically, forexample in a scanning-tunneling microscope. While pyrimidine bases canbe differentiated electronmicroscopically due to the methyl group at the5 position, this is not possible in the case of the purine bases adenineand guanine. It is not possible, therefore, to decode the base sequencesof nucleic acid molecules electronmicroscopically in a straightforwardmanner. However, if the nucleic acid molecule to be investigated nowcontain substituted 7-deazaguanine derivatives, for example, in place ofthe unmodified guanine bases, the substituted 7-deazaguanine bases canbe distinguished in the electron microscope from unsubstituted adeninebases (and, conversely, guanine bases can be distinguished fromsubstituted 7-deazaadenine bases). Consequently, the base sequences ofnucleic acids which contain 7-substituted 7-deazapurine bases can bedecoded by electron microscopy.

EXAMPLES

The compounds (1)-(25) named in the examples exhibit the followingstructural formulae.

The deoxytubercidin derivatives (1)-(3) (tubercidin=7-deazaadenosine)are prepared using the method described by Seela et al. [HelveticaChimica Acta, 1994, 77, 897-903)].

The corresponding ribonucleoside derivatives can be prepared in analogywith the following examples using a tubercidin derivative as thestarting compound.

Example 14-Benzoylamino-5-chloro-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(4)

1.14 g (4.0 mmol) of 5-chlorodeoxytubercidin (1) are evaporated twicewith dry pyridine and dissolved in 10 ml of dry pyridine, and thissolution is then stirred, at room temperature for 2 h, together with 5.2ml (40.6 mmol) of trimethylchlorosilane. 520 μl (4.1 mmol) of freshlydistilled benzoyl chloride are then added, and the mixture is stirred atroom temperature for a further 2 h. 4 ml of water and, after a further 5min, 8 ml of 25% aqueous ammonia are added dropwise while cooling withice. The mixture is stirred at room temperature for 30 min and thenevaporated to dryness. The residue is taken up in 20 ml of water, andthis solution is extracted three times with 30 ml of ethyl acetate oneach occasion. The organic phases are dried over Na₂SO₄ and evaporated,and the residue is chromatographed on silica gel (20×5 cm column,dichloromethane/methanol 9:1). 930 mg (2.4 mmol, 60%) of the compound(4) are obtained, as colorless crystals, from the more slowly migratingmain fraction after evaporating the solvent and recrystallizing theresidue from methanol/water: m.p. 190° C. TLC (silica gel,dichloromethane/methanol 9:1): R_(f)=0.4. UV (MeOH): λ_(max)=274 nm(5300), 305 nm (5600).

¹H-NMR ([D₆]DMSO): δ=2.31 (m, 2′α-H), 2.57 (m, 2′β-H), 3.58 (m, 2H,5′-H), 3.89 (m, 4′-H), 4.41 (m, 3′-H), 5.00 (t, J=5.0 Hz, 5′-OH), 5.33(d, J=5.3 Hz, 3′-OH), 6.72 (pt, J=6.75 Hz, 1′-H), 7.44-7.65 (m, 3H,meta- and para-H_(Bz)), 8.00 (s, 6-H), 8.05 (d, 2H, ortho-H_(Bz)), 8.72(s, 2-H), 11.2 (br, 4-NH).

C₁₈H₁₇ClN₄O₄ (388.8) Calc. C 55.61 H 4.41 N 14.41 Found C 55.71 H 4.54 N14.30

Example 24-Benzoylamino-5-bromo-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(5)

1.31 g (4.0 mmol) of 5-bromodeoxytubercidin (2) are evaporated twicewith dry pyridine and dissolved in 10 ml of dry pyridine, and thissolution is then stirred, at room temperature for 2 h, together with 5.2ml (40.6 mmol) of trimethylchlorosilane. 520 μl (4.1 mmol) of freshlydistilled benzoyl chloride are then added and the mixture is stirred atroom temperature for a further 2 h. 4 ml of water and, after a further 5min, 8 ml of 25% aqueous ammonia are added dropwise while cooling withice. The mixture is stirred at room temperature for 30 min and thenworked up in analogy with compound 14b. 1.2 g (2.8 mmol, 70%) ofcolorless crystals, of m.p. 198° C., are obtained after chromatographyon silica gel (20×5 cm column, dichloromethane/methanol 9:1),evaporation of the solvent and recrystallization from methanol/water.

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.4.

UV (MeOH): λ_(max)=276 nm (4600), 308 nm (4500).

¹H-NMR ([D₆] DMSO): δ=2.27 (m, 2′α-H), 2.50 (m, 2′β-H, overlapped byDMSO), 3.56 (m, 2H, 5′-H), 3.86 (m, 4′-H), 4.38 (m, 3′-H), 5.01 (t,J=5.0 Hz, 5′-OH), 5.34 (d, J=5.3 Hz, 3′-OH), 6.69 (pt, J=6.7 Hz, 1′-H),7.52-7.64 (m, 3H, meta- and para-H_(Bz))., 8.04 (d, 2H, ortho-H_(Bz)),8.04 (s, 6-H), 8.72 (s, 2-H), 11.0 (br, 4-NH).

C₁₈H₁₇BrN₄O₄ (433.3) Calc. C 49.90 H 3.96 N 12.93 Found C 50.04 H 4.10 N13.05

Example 34-Benzoylamino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-methyl-7H-pyrrolo[2,3-d]pyrimidine(6)

1.06 g (4.0 mmol) of 5-methyldeoxytubercidin (3) are reevaporated twicewith 20 ml of absolute pyridine on each occasion and dissolved in 10 mlof dry pyridine, and this solution is stirred, at room temperature, for2 h, together with 5.2 ml (40.6 mmol) of trimethylchlorosilane.

520 μl (4.1 mmol) of freshly distilled benzoyl chloride are then added,and the mixture is stirred at room temperature for a further 2 h. Theworking-up is carried out in analogy to that for compound (4), andchromatography then takes place on silica gel (20×5 cm column,dichloromethane/methanol 9:1). 1.1 g (2.9 mmol, 73%) of colorlesscrystals (compound 6), with a m.p. of 196° C., are obtained from themore slowly migrating main fraction after evaporating the solvent andrecrystallizing from methanol/water.

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.3.

UV (MeOH): λ_(max)=274 nm (7050), 309 nm (5500).

¹H-NMR ([D₆] DMSO): δ=2.09 (m, 2′α-H), 2.21 (s, 5-CH₃), 2.50 (m, 2′β-H,overlapped by DMSO), 3.53 (m, 2H, 5′-H), 3.83 (m, 4′-H), 4.36 (m, 3′-H),4.97 (t, J=5.0 Hz, 5′-OH), 5.32 (d, J=5.3 Hz, 3′-OH), 6.65 (pt, J=6.7Hz, 1′-H), 7.53 (s, 6-H), 7.53-7.66 (m, 3H, meta- and para-H_(Bz)), 8.05(d, 2H, ortho-H_(Bz)), 8.60 (s, 2-H), 10.95 (br, 4-NH).

C₁₉H₂₀N₄O₄ (368.4) Calc. C 61.95 H 5.47 N 15.21 Found C 62.08 H 5.65 N15.00

Example 45-Bromo-4-((1-dimethylamino)methylenelamino-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(7)

1.5 ml (8.75 mmol) of N,N-dimethylformamide diethyl acetal are added toa solution of 200 mg (0.61 mmol) of the compound (2) in 15 ml ofdimethylformamide, and the reaction solution is left to stir at roomtemperature for 2 h. It is then concentrated down to dryness, and theoily residue is reevaporated twice with toluene and twice with acetone.The crude product is adsorbed on silica gel and purified by columnchromatography (20×5 cm column, dichloromethane/methanol 9:1). Compound(7) is obtained, as colorless platelets (150 mg, 0.4 mmol, 65%): m.p.177° C., after concentrating the main fraction and recrystallizing theresidue from acetone/methanol 9:1.

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.65.

¹H-NMR ([D₆] DMSO): δ=2.20 (m, 2′α-H), 2.50 (m, 2′β-H, overlapped byDMSO), 3.18, 3.19 (2s, 2 N—CH₃), 3.54 (m, 2H, 5′-H), 3.86 (m, 4′-H),4.35 (m, 3′-H), 5.01 (t, J=5.5 Hz, 5′-OH), 5.26 (d, J=5.0 Hz, 3′-OH),6.57 (pt, J=6.9 Hz, 1′-H), 7.70 (s, 6-H), 8.34 (s, 2-H), 8.82 (s, N=CH).

C₁₄H₁₈BrN₅O₃ (384.2) Calc. C 43.77 H 4.72 N 18.23 Found C 43.92 H 4.80 N18.11

Example 54-Benzoylamino-5-chloro-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-7H-pyrrolo[2,3-d]pyrimidine(8)

500 mg (1.28 mmol) of compound (4) are evaporated twice with drypyridine and then dissolved in 20 ml of absolute pyridine. 650 mg (1.95mmol) of dimethoxytrityl chloride are added, and the mixture is stirredat room temperature for 1 h. It is then hydrolyzed with 10 ml of a 5%aqueous solution of NaHCO₃ and extracted twice with 25 ml ofdichloromethane on each occasion. After the combined organic phases havebeen dried over Na₂SO₄ chromatography takes place on silica gel (20×5 cmcolumn, dichloromethane/methanol 9:1). The residue which is obtainedafter inspissating the main zone yields, after evaporating with acetone,680 mg (0.99 mmol, 77%) of a yellowish foam. For the purification, thesubstance is dissolved in a little dichloromethane, and this solution isslowly added dropwise, while stirring vigorously, to a 200-fold excessof n-hexane. Compound (8) is isolated as a white, amorphous solid.

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.5.

¹H-NMR ([D₆] DMSO): δ=2.30 (m, 2′α-H), 2.50 (m, 2′β-H, overlapped byDMSO), 3.15 (m, 2H, 5′-H), 3.73 (s, 6H, 2 OCH₃), 3.98 (m, 4′-H), 4.42(m, 3′-H), 5.40 (d, J=5.0 Hz, 3′-OH), 6.69 (pt, J=6.7 Hz, 1′-H), 6.84(m, 4H, DMT), 7.2-7.8 (m, 12H, aromatic protons), 7.87 (s, 6-H), 8.06(d, 2H, ortho-H_(Bz)), 8.70 (s, 2-H), 11.0 (br, 4-NH).

C₃₉H₃₅ClN₄O₆ (691.2) Calc. C 67.77 H 5.10 N 8.11 Found C 67.70 H 5.05 N8.19

Example 64-Benzoylamino-5-bromo-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-7H-pyrrolo[2,3-d]pyrimidine(9)

500 mg (1.15 mol) of compound (5) are evaporated twice with dry pyridineand subsequently dissolved in 20 ml of absolute pyridine. 585 mg (1.75mmol) of dimethoxytrityl chloride are added, and the mixture is stirredat room temperature for 1 h. It is then hydrolyzed with 10 ml of a 5%aqueous solution of NaHCO₃ and extracted twice with 25 ml ofdichloromethane on each occasion. After the combined organic phases havebeen dried over Na₂SO₄, chromatography takes place on silica gel (20×5cm column, dichloromethane/methanol 9:1). The residue which is obtainedafter inspissating the main zone yields, after evaporating with acetone,620 mg (0.93 mmol, 80%) of a yellowish foam. For the purification, thesubstance is dissolved in a little dichloromethane and this solution isslowly added dropwise, while stirring vigorously, to a 200-fold excessof n-hexane. Compound (9) precipitates out as a white amorphous solidand is filtered off with suction.

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.55.

¹H-NMR ([D₆] DMSO): δ=2.30 (m, 2′α-H), 2.50 (m, 2′β-H, overlapped byDMSO), 3.15 (m, 2H, 5′-H), 3.73 (s, 6H, 2 OCH₃), 3.98 (m, 4′-H), 4.42(m, 3′-H), 5.40 (d, J=5.0 Hz, 3′-OH), 6.69 (pt, J=6.7 Hz, 1′-H), 6.84(m, 4H, DMT), 7.2-7.8 (m, 12H, aromatic protons), 7.87 (s, 6-H), 8.06(d, 2H, ortho-H_(Bz)), 8.70 (s, 2-H), 11.0 (br, 4-NH).

C₃₉H₃₅BrN₄O₆ (735.6) Calc. C 63.68 H 4.79 N 7.62 Found C 63.85 H 4.67 N7.52

Example 7 4-Benzoylamino-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-5-methyl-7H-pyrrolo[2,3-d]pyrimidine(10)

500 mg (1.36 mmol) of compound (6) are evaporated twice with 20 ml ofdry pyridine on each occasion and dissolved in 20 ml of absolutepyridine, and this solution is stirred, at room temperature for 1 h,together with 690 mg (2.1 mmol) of dimethoxytrityl chloride. The mixtureis worked up in an analogous manner to that employed for compound (8),and chromatographed on silica gel (20×5 cm column,dichloromethane/methanol 9:1). 720 mg (1.05 mmol, 77%) of the completelyprotected compound (10) are obtained, as a yellowish foam, from the mainzone. Purification, by reprecipitating from n-hexane, yields acolorless, amorphous solid.

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.5.

¹H-NMR ([D₆] DMSO): δ=2.08 (s, 5-CH₃), 2.30 (m, 2′α-H), 2.50 (m, 2′β-H,overlapped by DMSO), 3.10 (m, 2H, 5′-H), 3.73 (s, 6H, 2 OCH₃), 3.97 (m,4′-H), 4.44 (m, 3′-H), 5.39 (d, J=5.0 Hz, 3′-OH), 6.67 (pt, J=6.7 Hz,1′-H), 6.85 (m, 4H, DMT), 7.2-7.8 (m, 12H, aromatic protons), 7.58 (s,6-H), 8.06 (d, 2H, ortho-H_(Bz)), 8.60 (s, 2-H), 10.95 (br, 4-NH).

C₄₀H₃₈N₄O₆ (670.8) Calc. C 71.63 H 5.71 N 8.35 Found C 71.48 H 5.71 N8.36

Example 84-Benzoylamino-5-chloro-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-7H-pyrrolo[2,3-d]pyrimidine-3′-(triethylammoniumphosphonate) (11)

840 mg (12.0 mmol) of 1,2,4-1H-triazole are added, at room temperatureand under an argon atmosphere, to a solution of 315 μl (3.7 mmol) ofphosphorus trichloride and 4.1 ml (37.0 mmol) of N-methylmorpholine in40 ml of absolute dichloromethane. After the mixture has been stirredfor 30 minutes, it is cooled down to 0° C. and a solution of 500 mg(0.74 mmol) of the fully protected nucleoside (8) in 10 ml of drydichloromethane is added dropwise over a period of 10 min. The mixtureis left to stir at room temperature for a further 10 min, and 30 ml of 1M triethylammonium bicarbonate buffer (TBC, pH=7.5) are then added. Thephases are separated and the aqueous phase is extracted several timeswith CH₂Cl₂; the combined organic phases are then dried over Na₂SO₄. Thesolvent is evaporated off and the remaining foam is chromatographed onsilica gel (20×5 cm column, 0.5 l dichloromethane/Et₃N 98:2, and thendichloromethane/methanol/Et₃N 88:10:2). After the main zone has beenconcentrated, the residue is taken up in 50 ml of dichloromethane andthis solution is extracted five times with 25 ml of 0.1 M TBC buffer oneach occasion. 445 mg (0.52 mmol, 70%) of the phosphonate (11) areobtained, as a colorless foam, after drying the organic phase overNa₂SO₄ and evaporating off the solvent. For the further purification,this foam is reprecipitated from n-hexane in an analogous manner to thatused for the fully protected nucleoside (8).

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.6.

¹H-NMR ([D₆] DMSO): δ=1.16 (t, 9H, (CH₃CH₂)₃N), 2.50 (m, 2′α-H,overlapped by DMSO), 2.74 (m, 2′β-H), 3.00 (q, 6H, (CH₃CH₂)₃N), 3.33 (m,2H, 5′-H), 3.72 (s, 6H, 2 OCH₃), 4.15 (m, 4′-H), 4.78 (m, 3′-H), 6.66(d, J=585.8 Hz, P—H), 6.69 (pt, J=7.8 Hz, 1′-H), 6.84 (m, 4H, DMT),7.2-7.7 (m, 12H, aromatic protons), 7.79 (s, 6-H), 8.04 (d, 2H,ortho-H_(Bz)), 8.69 (s, 2-H), 10.6 (br, 4-NH).

³¹P-NMR ([D₆] DMSO): δ=1.16 ppm (dd, ¹J(PH)=588 Hz, ³J(PH)=8.6 Hz.C₄₅H₅₁ClN₅O₈P (900.8)

Example 94-Benzoylamino-5-bromo-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-7H-pyrrolo[2,3-d]pyrimidine-3′-(triethylammoniumphosphonate) (12)

770 mg (11.0 mmol) of 1,2,4-1H-triazole are added, at room temperatureand under an argon atmosphere, to a solution of 290 μl (3.4 mmol) ofphosphorus trichloride and 3.8 ml (34.0 mmol) of N-methylmorpholine in30 ml of absolute dichloromethane. After the mixture has been stirredfor 30 minutes, it is cooled down to 0° C. and a solution of 500 mg(0.68 mmol) of the completely protected nucleoside (9) in 10 ml of drydichloromethane is added dropwise within the space of 10 min. Themixture is left to stir at room temperature for a further 10 min and 30ml of 1 M triethylammonium bicarbonate buffer (TBC, pH=7.5) are thenadded. The phases are separated, the aqueous phase is extracted severaltimes with CH₂Cl₂, and the combined organic phases are dried overNa₂SO₄. The solvent is evaporated off and the remaining foam ischromatographed on silica gel (20×5 cm column, 0.5 l ofdichloromethane/Et₃N, 98:2, and, after that,dichloromethane/methanol/Et₃N, 88:10:2). After the main zone has beenconcentrated, the residue is taken up in 50 ml of dichloromethane andthis solution is extracted five times with 25 ml of 0.1 M TBC buffer oneach occasion. 410 mg (0.46 mmol, 67%) of the phosphonate (12) areobtained as a colorless foam after the organic phase has been dried overNa₂SO₄ and the solvent has been evaporated off. For the furtherpurification, this foam can be reprecipitated from n-hexane in ananalogous manner to that for the completely protected nucleoside (9).

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.7.

¹H-NMR ([D₆] DMSO): δ=1.16 (t, 9H, (CH₃CH₂)₃N), 2.50 (m, 2′α-H,overlapped by DMSO), 2.78 (m, 2′β-H), 3.00 (q, 6H, (CH₃CH₂)₃N), 3.22 (m,2H, 5′-H), 3.73 (s, 6H, 2 OCH₃), 4.17 (m, 4′-H), 4.82 (m, 3′-H), 6.68(d, J=588.5 Hz, P—H), 6.69 (pt, J=6.7 Hz, 1′-H), 6.90 (m, 4H, DMT),7.2-7.8 (m, 12H, aromatic protons), 7.86 (s, 6-H), 8.07 (d, 2H,ortho-H_(Bz)), 8.70 (s, 2-H), 11.05 (br, 4-NH).

³¹P-NMR ([D₆] DMSO): δ=1.16 ppm (dd, ¹J(PH)=588 Hz, ³J(PH)=8.6 Hz).C₄₅H₅₁BrN₅O₈P (900.8)

Example 10 4-Benzoylamino-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-5-methyl-7H-pyrrolo[2,3-d]pyrimidine-3′-(triethylammoniumphosphonate) (13)

0.78 g (11.3 mmol) of 1,2,4-H-triazole are added, at room temperatureand under an argon atmosphere, to a solution of 25 ml of absolutedichloromethane, 290 μl (3.4 mmol) of phosphorus trichloride and 3.44 g(34.0 mmol) of N-methylmorpholine. After it has been stirred for 30minutes, the reaction solution is cooled down to 0° C. and a solution of500 mg (0.75 mmol) of the fully protected nucleoside (10) in 15 ml ofdichloromethane is added within the space of 10 min. The mixture isallowed to stir at room temperature for a further 20 min and is thenhydrolyzed with 1 M triethylammonium bicarbonate buffer (TBC, pH=7.5).After the phases have been separated, the aqueous phase has beenextracted three times with 20 ml of dichloromethane on each occasion,and the organic phase has been dried and concentrated by evaporation,the residue is chromatographed on silica gel (20×5 cm column, 0.5 l ofdichloromethane/Et₃N, 98:2, and, after that,dichloromethane/methanol/Et₃N, 88:10:2). The main zone is inspissatedand the residue is taken up in 50 ml of dichloromethane and thissolution is extracted several times with 0.1 M TBC buffer. 440 mg (0.53mmol, 70%) of the compound (13) are obtained as a colorless foam afterdrying the organic phase over Na₂SO₄ and evaporating off the solvent.

TLC (silica gel, dichloromethane/methanol 9:1): R_(f)=0.65.

¹H-NMR ([D₆] DMSO): δ=1.16 (t, 9H, (CH₃CH₂)₃N), 2.09 (s, 5-CH₃), 2.24(m, 2′α-H), 2.67 (m, 2′β-H), 3.00 (q, 6H, (CH₃CH₂)₃N), 3.20 (m, 2H,5′-H), 3.73 (s, 6H, 2 OCH₃), 4.13 (m, 4′-H), 4.83 (m, 3′-H), 6.65 (pt,J=6.5 Hz, 1′-H), 6.68 (d, J=588.5 Hz, P—H), 6.85 (m, 4H, DMT), 7.2-7.6(m, 12H, aromatic protons), 7.58 (s, 6-H), 8.05 (d, 2H, ortho-H_(Bz)),8.60 (s, 2-H), 10.98 (br, 4-NH).

³¹P-NMR ([D₆] DMSO): δ=1.08 ppm (dd, ¹J(PH)=577 Hz, ³J(PH)=8.9 Hz).C₄₆H₅₄N₅O₈P (835.8)

Example 114-Amino-5-bromo-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine5′-O-triphosphate, triethylammonium Salt

Compound (2) (33 mg, 0.1 mmol) is dissolved, together with1,8-bis(dimethylamino)naphthalene (33 mg, 0.15 mmol), in trimethylphosphate (0.25 ml) while warming gently. After the solution has beencooled down to 0° C., freshly distilled POCl₃ (12 μl, 0.13 mmol) isadded. The reaction mixture is maintained at 4° C. for 4 h and asolution comprising tri-n-butylanmonium diphosphate (0.5 m in DMF, 1 ml)and tri-n-butylamine (100 μl, 0.42 mmol) is then added. After themixture has been stirred at 0° C. for 3 min, 1 M TBC buffer (10 ml) isadded and the whole is evaporated to dryness. The residue ischromatographed on DEAD Sephadex (1.5×20 cm column, HCO₃ ⁻ form). Afterwashing the column with approximately 500 ml of H₂O, chromatography tookplace using a linear gradient of H₂O/0.9 M TBC buffer (1 l in eachcase). During this procedure, the triphosphate (0.019 mM, 20%) isobtained at approximately 0.5 M TBC buffer.

TLC (silica gel, i-propanol/H₂O/NH₃, 3:1:1): R_(f)=0.2. UV (H₂O):λ_(max)=269 nm.

³¹P-NMR (0.1 M tris-HCl, pH 8.0, 100 mM EDTA/D₂O): −11.87 (d, J=20.2,P_(y)); −10.98 (td, J=20.0 and 6.0, P_(α)); −23.06 (t, J=20.2, P_(β)).

Example 124-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine(14) (5-iodo-2′-deoxytubercidin)

25% aqueous ammonia is added to 1.0 g (2.5 mmol) of4-chloro-7-[2-deoxy-3,5-di-O-(4-toluoyl)-β-D-erythropentofuranosyl]-5-iodo-7H-pyrrolo[2,3-d]pyrimidinewhich is dissolved in 80 ml of dioxane (80 ml). The mixture is stirredin a steel cylinder at 110° C. for 48 h. After the solvent has beenevaporated off, the concentrated residue is chromatographed on silicagel (20×5 cm column, solvent B). Colorless crystals from MeOH (0.75 g,2.0 mmol, 45%). M.p. 194° C. TLC: R_(f) 0.4 (CH₂Cl₂/MeOH, 9:1). UV(MeOH) 283 nm (5 800).

¹H-NMR (D₆-DMSO): 2.16 (m, H-2′α), 2.46 (m, H-2′_(β), overlapped byDMSO), 3.54 (m, 2-H, H-5′), 3.81 (m, H-4′), 4.33 (m, H-3′), 5.00 (t,J=5.1 Hz, 5′-OH), 5.23 (d, J=5.1 Hz, 3′-OH), 6.49 (pt, J=6.7 Hz, H-1′),6.65 (br, NH₂), 7.65 (s, H-6), 8.10 (s, H-2).

¹³C-NMR (D₆-DMSO) 157.3 (C-4), 152.0 (C-2), 149.8 (C-7a), 126.9 (C-6),103.2 (C-4a), 87.5 (C-4′), 83.0 (C-1′), 71.0 (C-3′), 62.0 (C-5′), 51.9(C-5), 39.8 (C-2′).

Anal. calculated for C₁₁H₁₃IN₄O₃: C 35.13, H 3.48, N 14.90; found: C35.33, H 3.69, N 15.01.

Example 13 Cross-Coupling Reaction of 5-iodo-2′-deoxytubercidin (14)

5-Iodo-2′-deoxytubercidin (14) (200 mg, 0.532 mmol) and copper(I) iodide(10 mg, 10 mol %) are suspended in 3 ml of dry DMF, which has beenpreviously flushed with argon, and alkyne (6-15 eq.), dry triethylamine(108 mg, 1.06 mmol, 2 eq.) and tetrakis(triphenylphosphine)palladium (0)(30.75 mg, 0.027 mmol, 5 mol %) are added to this mixture.

Within a few hours, the mixture turns into a clear yellow solution. Thereaction is continued until the starting compounds are used up(monitoring by thin layer chromatography). The reaction mixture is thendiluted with 5 ml of methanol/dichloromethane (1:1), and Dowex 1×8(100-200 mesh; 500 mg, bicarbonate form) is added. Once the gasformation has ceased, after 15 minutes of stirring, the reaction mixtureis stirred for a further 30 minutes. It is then filtered and the matrixis washed with methanol/dichloromethane (1:1).

The filtrates are combined and dried. The dried residue is immediatelychromatographed on a silica gel column (25 g) using dichloromethanehaving an increasing content of methanol (10, 15, 20%). The substituted2′-deoxytubercidin derivative is obtained after evaporating the mainfraction.

Example 14 4-Benzoylamino-5-(1-propynyl-3-trifluoroacetamide)-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-7H-pyrrolo[2,3-d]-pyrimidine-3′-(triethylammoniumphosphonate) (15)

a) 7-Deaza-2′-deoxy-7-(1-propynyl-3-trifluoroacetamide)adenosine

5-Iodo-2′-deoxytubercidin (14) from Example 12 is coupled, under theconditions described in Example 13 and over a period of 9 h, toN-propargyltrifluoroacetamide. The following quantities are employed:5-iodo-2′-deoxytubercidin (14) (200 mg, 0.532 mmol), copper(I) iodide(5.0 mg, 0.0236 mmol, 5 mol %), DMF (3 ml),N-propargyltrifluoroacetamide (482 mg, 3.2 mmol, 6 eq.), triethylamine(108 mg, 1.06 mmol, 2 eq.) and tetrakis(triphenylphosphine)palladium (0)(61.5 mg, 0.0532 mmol, 10 mol %). Following chromatography, the solid isrecrystallized from ethyl acetate: pale yellow crystals (70 mg, 0.176mmol, 33%). M.p. 187-188° C. TLC: R_(f) 0.30 (CH₂Cl₂/MeOH, 9:1). UV(MeOH) 237 (14 400), 279 (14 200).

¹H-NMR (D₆-DMSO) 10.07 (s, 1H, NHTFA), 8.12 (s, 1H, H-2), 7.76 (s, 1H,H-6), 6.79 (broad s, 2H, NH₂), 6.49 (pt, 1H, H-1′, J=6.6 Hz), 5.25 (d,1H, 3′-OH, J=3.0 Hz), 5.05 (t, 1H, 5′-OH, J=4.5 Hz), 4.35 (m, 1H, H-3′),4.32 (d, 2H, CH₂, J=4.2 Hz), 3.84 (m, 1H, H-4′), 3.56 (m, 2H, H-5′),2.47 (m, 1H, H-2′β), 2.19 (m, 1H, H-2′α).

¹³C-NMR (D₆-DMSO): 157.4 (C-4), 156.4 and 156.1 (C═O), 152.7 (C-2),149.2 (C-7a), 126.5 (C-6), 116.8 and 114.6 (CF₃), 102.2 (C-4a), 94.0(C-5), 87.5 (C-4′), 86.7 and 76.2 (C═C), 83.2 (C-1′), 70.9 (C-3′), 61.8(C-5′), 39.6 (C-2′, overlapped by DMSO), 29.9 (CH₂).

Anal. calculated for C₁₆H₁₆F₃N₅O₄: C 48.13, H 4.04, N 17.54; found: C48.26, H 4.13, N 17.58.

b)4-Benzoylamino-5-(1-propynyl-3-trifluoroacetamide)-7-[(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]-pyrimidine

The benzoylamino protective group is inserted into7-deaza-2′-deoxy-7-(1-propynyl-3-trifluoroacetamide)adenosine in analogywith Example 1.

c)4-Benzoylamino-5-(1-propynyl-3-trifluoroacetamide)-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-7H,-pyrrolo[2,3-d]-pyrimidine

The Dmt-hydroxyl protective group is inserted into4-benzoylamino-5-(1-propynyl-3-trifluoroacetamide)-7-[(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]-pyrimidinein analogy with Example 5.

d) The title compound (15) is prepared from4-benzoylamino-5-(1-propynyl-3-trifluoroacetamide)-7-((2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-7H-pyrrolo[2,3-d]pyrimidinein analogy with Example 8.

Example 154-Benzoylamino-5-(1-pentynyltrifluoroacetamide)-7-[(2-deoxy-β-D-erythropentofuranosyl)-5′-O-(4,4′-dimethoxytriphenylmethyl)]-7H-pyrrolo[2,3-d]-pyrimidine-3′-(triethylammonium phosphonate) (16)

a) 7-Deaza-2′-deoxy-7-(1-pentynyltrifluoroacetamide)adenosine

5-Iodo-2′-deoxytubercidin (14) from Example 12 is coupled, under theconditions described in Example 13 and over a period of 48 h, to5-trifluoroacetamide-1-pentyne. The following quantities are employed:5-iodo-2′-deoxytubercidin (200 mg, 0.532 mmol), copper(I) iodide (5 mg,0.0236 mmol, 5 mol %), DMF (3 ml), 5-trifluoroacetamide-1-pentyne (953mg, 5.32 mmol, 10 eq.), triethylamine (108 mg, 1.06 mmol, 2 eq.) andtetrakis(triphenylphosphine)palladium (0) (61.5 mg, 0.0532 mmol, 10 mol%). Following chromatography, a weakly yellowish oily residue (84.1 mg,0.197 mmol, 37%) is obtained from the liquid by crystallization. M.p.51-52° C. TLC: R_(f) 0.35 (CH₂Cl₂/MeOH, 9:1). UV (MeOH) max=239 (14300), 280 (10 900).

¹H-NMR (D₆-DMSO): 8.08 (s, 1H, H-2), 7.64 (s, 1H, H-6), 6.46 (pt. 1H,H-1′, J=6.9 Hz), 4.32 (m, 1H, H-3′), 3.80 (m, 1H, H-4′), 3.59-3.28(several m, 5H, H-5′, CH ₂—CH₂—CH ₂—N), 2.47 (m, 1H, H-2′β), 2.17 (m,1H, H-2′α), 1.76 (quintet, 2H, CH₂—CH ₂—CH₂—N). ¹³C-NMR (D₆-DMSO): 157.6(C-4), 156.5 and 156.2 (C═O), 152.7 (C-2), 149.2 (C-7a), 125.7 (C-6),118.5 and 114.0 (CF₃), 102.3 (C-4a), 95.5 (C-5), 91.6 (C═C, 1″), 87.6(C-4′), 83.2 (C-1′), 74.0 (C═C, 2″), 71.0 (C-3′), 62.0 (C-5′), 39.6 and38.6 (C-2′ and CH₂, overlapped by DMSO), 27.7 (CH₂), 16.6 (CH₂).

Anal. calculated for C₁₈H₂₀N₅O₄F₃: C 50.59, H 4.72, N 16.39; found: C50.65, H 4.82, N 16.32.

The title compound (16) is obtained from7-deaza-2′-deoxy-7-(1-pentynyltrifluoroacetamide)adenosine in analogywith Examples 14b), 14c) and 14d).

Example 16 2′-Deoxy-7-deaza-7-(2-carboxyethenyl)adenosine (17).

5-Iodo-2′-deoxytubercidin (14) from Example 12 is coupled, under theconditions described in Example 13 and over a period of 65 h, to methylacrylate. The following quantities are employed:5-iodo-2′-deoxytubercidin (200 mg, 0.532 mmol), copper(I) iodide (5 mg,0.0236 mmol, 5 mol %), DMF (3 ml), triethylamine (108 mg, 1.06 mmol, 2eq.), methyl acrylate (686 mg, 8.0 mmol, 15 eq.) andtetrakis(triphenylphosphine)palladium (0) (61.5 mg, 0.0532 mmol, 10 mol%). A pale yellow foam is obtained after chromatographic purification,and 71.1 mg of solid substance (40%) are obtained after washing withdichloromethane. M.p. 101-102° C. TLC: R_(f) 0.40 (CH₂Cl₂/MeOH, 9:1). UV(MeOH) max=268.0 (13 500), 324.8 (11 900).

¹H-NMR (D₆-DMSO): 8.11 (2s, 2H, H-2 and H-6), 7.94 (d, 1H, H-1″, J=15.6Hz), 6.86 (s, 2H, NH₂), 6.51 (pt, 1H, H-1′, J=6.6 Hz), 6.4 (d, 1H, H-2″,J=15.6 Hz), 5.26 (d, 1H, 3′-OH, J=3.6 Hz), 5.04 (t, 1H, 5′-OH, J=5.1Hz), 4.36 (m, 1H, H-3′), 3.83 (m, 1H, H-4′), 3.70 (s, 3H, OCH₃), 3.55(m, 1H, H-5′), 2.45 (m, 1H, H-2′β), 2.22 (m, 1H, H-2′α).

¹³C-NMR (D₆-DMSO): 166.9 (C═O ), 158.0 (C-4), 152.1 (C-2), 151.2 (C-7a),137.4 (CH—C═O), 123.7 (C-6), 115.5 (CH═CH—C═O), 111.5 (C-5), 101.0(C-4a), 87.6 (C-4′), 83.2 (C-1′), 70.9 (C-3′), 62.0 (C-5′), 51.2 (OCH₃),39.6 (C-2′, overlapped by DMSO).

Anal. calculated for C₁₅H₁₈N₄O₅: C 53.89, H 5.43, N 16.76; found: C53.79, H 5.56, N 16.74.

Example 177-[2-Deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-4-methoxy-2-[(formyl)amino]-7H-pyrrolo[2,3-d]-pyrimidine(18).

7-(2-Deoxy-β-D-erythropentofuranosyl)-4-methoxy-2-[(formyl)amino]-7H-pyrrolo[2,3-d]pyrimidine(1.0 g, 3.3 mmol) [F. Seela, H. Driller, Nucleosides, Nucleotides 1989,8, 1-21] in acetonitrile (20 ml) was stirred, at room temperature for 15hours, together with isobutyric anhydride (33 mmol) in the presence oftriethylamine (23 mmol). The solvent is evaporated off and the residueis reevaporated with methanol. It is then chromatographed in the eluentmethylene chloride/acetone (95:5), and the main zone is isolated and theconstituent compound is recrystallized from cyclohexane. 1.26 g (89%) ofa colorless solid.

¹H-NMR ([D₆] DMSO), δ: 1.05-1.14 (m, 4CH₃), 2.60, 2.90 (m, CH and2′-Ha,b), 4.02 (S, OCH₃), 4.16 (m, 5′-H), 4.26 (m, 4′-H), 5.35 (m,3′-H), 6.47 (m, 1′-H), 6.52 (d, 5-H), 7.39 (d, 6-H), 9.44 (d, NH), 10.71(d, HCO).

Example 185-Bromo-7-[2-deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-4-methoxy-2-[(formyl)amino]-7H-pyrrolo[2,3-d]pyrimidine(19).

A solution of compound 18 (10.1 mmol) in dimethylformamide was stirred,at room temperature for 1 hour, together with N-bromosuccinimide (10.1mmol). A few drops of 5% aqueous NaHCO₃ are added to the solution, inorder to buffer it, and methylene chloride is then added. The organicphase is shaken with water, separated, dried over sodium sulfate andevaporated. Chromatography of the residue on a silica gel column in theeluent dichloromethane/acetone (95:5) results in two zones. Theevaporation residue from the slowly migrating main zone yields colorless(19) (75%) as a solid.

¹H-NMR ([D₆] DMSO), δ: 1.05-1.14 (m, 4CH₃), 2.60, 2.88 (m, CH and2′-Ha,b), 4.04 (s, OCH₃), 4.16 (m, 5′-H), 4.22 (m, 4′-H), 5.34 (m,3′-H), 6.46 (m, 1′-H), 7.60 (s, 6-H), 9.43 (s, NH), 10.86 (s, HCO).

A colorless solid, which was characterized as5,6-dibromo-7-[2-deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-4-methoxy-2-[(formyl)amino]-7H-pyrrolo[2,3-d]pyrimidine,is obtained from the rapidly migrating subsidiary zone from theabovementioned reaction.

¹H-NMR ([D₆] DMSO), δ: 0.99-1.13 (m, 4CH₃), 2.59, 3.58 (m, CH and2′-Ha,b), 4.03 (s, OCH₃), 4.16 (m, 5′-H), 4.30 (m, 4′-H), 5.56 (m,3′-H), 6.39 (m, 1′-H), 9.42 (s, NH), 10.91 (s, HCO).

Example 195-Chloro-7-[2-deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-4-methoxy-2-amino-7H-pyrrolo-[2,3-d]pyrimidine (20).

Using N-chlorosuccinimide, the substance was prepared and worked up inanalogy with compound (19). The halogenation reaction time was 8 hours.Acetonitrile/DMF (4:1) was used as the solvent. Colorless solid (70%).

¹H-NMR ([D₆] DMSO), δ: 1.07-1.13 (m, 4CH₃), 2.59, 2.74 (m, CH and2′-Ha,b), 3.93 (s, OCH₃), 4.15 (m, 5′-H), 4.21 (m, 4′-H), 5.25 (m,3′-H), 6.39 (m, 1′-H), 6.47 (5, NH₂), 7.20 (s, 6-H).

The compound5,6-dichloro-7-[2-deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-4-methoxy-2-amino-7H-pyrrolo[2,3-d]pyrimidine,in the form of a colorless solid, was obtained an a by-product.

¹H-NMR ([D₆] DMSO), δ: 1.03-1.13 (m, 4CH₃), 2.58, 3.40 (m, CH and2′-Ha,b), 3.93 (s, OCH₃), 4.16 (m, 5′-H), 4.37 (m, 4′-H), 5.44 (m,3′-H), 6.37 (m, 1′-H), 6.58 (s, NH₂).

Example 202-Amino-7-(2′-deoxy-β-erythropentofuranosyl)-3,7-dihydro-5-bromo-4-pyrrolo[2,3-d]pyrimidin-4-one(21)

Compound (19) is reacted using a known method [F. Seela, B. Westermann,U. Bindig, J. Chem. Soc. Perkin Trans I 1988, 699]. 500 mg of compound(19) are heated under reflux for 3 hours in 200 ml of 2 N NaOH. Thecooled solution is neutralized with glacial acetic acid, the inorganicresidue is filtered off, and the aqueous phase is evaporated.Recrystallization from water results in colorless crystals of (21).

Example 212-Amino-7-(2′-deoxy-β-erythropentofuranosyl)-3,7-dihydro-5-bromo-4-pyrrolo[2,3-d]pyrimidin-4-one(22)

Compound (22) is prepared in analogy with the method described inExample 20 and proceeding from compound (20). Recrystallization fromwater results in colorless crystals of (22).

Example 224-Amino-7-[2-deoxy-β-D-erythropentofuranosyl]-5-trimethylsilylethynyl-7H-pyrrolo[2,3-d]pyrimidine(23)

Compound (23) is prepared from trimethylsilylacetylene in accordancewith the general protocol for the cross-coupling reaction in Example 13.Colorless solid. Yield 54%.

Calc. C 55.47, H 6.40, N 16.17; found C 55.57, H 6.53, N 16.20.

¹H-NMR (DMSO): 8.12 (s, 1H, H-2), 7.80 (s, 1H, H-6), 6.76 (broad, 2H,NH₂), 6.47 (“t”, 1H, H-1′, J=6.7 Hz), 5.23 (d, 1H, 3′-OH, J=3.3 Hz),5.07 (t, 1H, 5′-OH), 4.33 (m, 1H, H-3′), 3.87 (m, 1H, H-4′), 3.54 (m,2H, H-5′), 2.46 (m, 1H, H-2′), 2.17 (m, 1H, H-2′), 0.73 (s, 9H, Me).

Example 234-Amino-7-[2-deoxy-β-D-erythropentofuranosyl]-5-ethynyl-7H-pyrrolo[2,3-d]pyrimidine(24)

200 mg of compound (23) are dissolved in 20 ml of MeOH. Adding 8 mg ofK₂CO₃ results in hydrolysis after 1 h of stirring. After the solutionhas been subjected to rotary evaporation, the residue is chromatographedon silica gel in the eluent methylene chloride/MeOH (8:1).Recrystallization from MeOH results in colorless crystals (73%).

Calc. C 56.93, H 5.15, N 20.43; found C 56.77, H 5.71, N 20.42.

¹H-NMR (DMSO): 8.13 (s, 1H, H-2), 7.81 (s, 1H, H-6), 6.65 (broad, 2H,NH₂), 6.49 (t, 1H, H-1′), 5.25 (m, 1H, 3′-OH), 5.05 (m, 1H, 5′-OH), 4.36(m, 1H, H-3′), 4.26 (s, 1H, ethyne), 3.84 (s, 1H, H-4′), 3.56 (m, 2H,H-5′), 2.47 (m, 1H, H-2′), 2.21 (m, 1H, H-2′).

Example 244-Amino-7-[2-deoxy-β-D-erythropentofuranosyl]-5-hexynyl-7H-pyrrolo[2,3-d]pyrimidine(25)

Compound (25) is prepared in accordance with the general protocol forthe cross-coupling reaction (Ex. 13) and using 1-hexyne.Recrystallization from MeOH results in colorless crystals (yield: 48%).

Calc. C 61.80, H 6.71, N 19.96; found C 61.68, H 6.60, N 16.90

¹H-NMR (DMSO): 8.31 (s, 1H, H-2), 7.65 (8, 1H, H-6), 6.65 (broad, 2H,NH₂), 6.49 (“t”, 1H, H-1′), 5.24 (m, 1H, 3′-OH), 5.05 (m, 1H, 5′-OH),4.50 (m, 1H, 3′-H), 3.84 (m, 1H, H-4′), 3.56 (m, 2H, H-5′), 2.48 (m, 2H,CH₂C═), 2.46 (m, 1H, H-2′), 2.18 (m, 1H, H-2′), 1.54 (m, 2H, CH₂), 1.43(m, 2H, CH₂), =0.93 (m, 2H, CH₃).

Example 252-Amino-6-chloro-7-[2-deoxy-3,5-di-O-acetyl-β-D-erythropentofuranosyl]-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine.

36.6 mg (0.27 mmol) of N-chlorosuccinimide are added to a solution of 50mg (0.14 mmol) of2-amino-7-(2-deoxy-3,5-di-O-acetyl-β-D-erythropentofuranosyl)-4-methoxy-7H-pyrrolo[2,3-d]pyrimidinein 3 ml of dichloromethane, and the mixture is stirred at roomtemperature for 12 h. The solvent is stripped off and the residue ischromatographed on silica gel in CH₂Cl₂/acetone (9:1). 30 mg (54%) of acolorless foam are obtained from the slowly migrating main zone.

¹H-NMR D₆ (DMSO): 1.99 (s, 3H, CH₃), 2.09 (s, 3H, CH₃), 2.37 (m, 1H,H-2′b), 3.93 (s, 3H, OCH₃), 4.18 (m, 2H, H-5′), 4.43 (m, 1H, H-4), 5.44(m, 1H, H-3′), 6.38 (m, 1H, H-1′), 6.42 (s, 1H, H-5).

¹³C-NMR (DMSO): 20.48 (CH₃), 20.76 (CH₃), 33.78 (C-2′), 52.99 (OCH₃),63.47 (C-5′), 74.31 (C-3′), 80.91 (C-4′), 82.95 (C-1′), 96.45 (C-4a),98.65 (C-5), 118.12 (C-6), 153.69 (C-7a), 159.25 (C-2), 162.16 (C-4),169.98 (C═O), 170.09 (C═O).

Example 262-Amino-5,6-dichloro-7-[2-deoxy-3,5-di-O-acetyl-β-D-erythropentofuranosyl]-4-methoxy-7H-pyrrolo[2,3-d]-pyrimidine.

The more rapidly migrating zone yields a colorless foam. 6 mg (9.9%).

¹H-NMR (DMSO): 1.98 (s, 3H, CH₃), 2.07 (s, 3H, CH₃), 2.24 (m, 1H,H-2′b), 2.73 (m, 1H, H-2′a), 3.94 (s, 3H, OCH₃), 4.14 (m, 2H, H-5′),4.39 (m, 1H, H-4), 5.40 (m, 1H, H-3′), 6.39 (m, 1H, H-1′), 6.59 (s, 2H,NH₂).

¹³C-NMR (DMSO): 20.47 (CH₃), 20.75 (CH₃), 34.06 (C-2′), 53.34 (OCH₃),63.41 (C-5′), 74.09 (C-3′), 81.01 (C-4′), 83.26 (C-1′), 94.59 (C-4a),102.08 (C-5), 115.16 (C-6), 151.94 (C-7a), 159.59 (C-2), 162.08 (C-4),169.97 (C═O), 170.07 (C═O).

UV (MeOH): λ_(max) (ε): 290 nm (8500), 264 nm (13100), 226 nm (22700).

Example 277-(2-Deoxy-β-D-erythropentofuranosyl)-4-[(dimethylamino)methylidene]amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidine(26)

A solution of 5-iodo-2′-deoxytubercidin (14) (400 mg, 1.06 mmol) inmethanol (20 ml) is stirred, at 40° C. for 2 h, together withN,N-dimethylformamide dimethyl acetyl (2.0 g, 16.8 mmol). After thesolvent has been evaporated off, the residue is purified by flashchromatography (FC) (column: 20×5 cm, CH₂Cl₂/MeOH, 9:1). The main zoneyields a colorless foam (389 mg, 85%). TLC (thin layer chromatography,silica gel, CH₂Cl₂/MeOH, 9:1): R_(f) 0.46. UV (MeOH): max=229 nm(17400), 277 nm (10400), 323 nm (19000). ¹H-NMR (D₆-DMSO): 2.18 (m, 1H,H_(α)-2′); 2.47 (m, 1H, H_(β)-2′, overlapped by DMSO); 3.18, 3.22 (2s,6H, N(CH₃)₂); 3.54 (m, 2H, H-5′); 3.81 (m, 1H, H-4′); 4.32 (m, 1H,H-3′); 5.00 (t, J=5.4 Hz, 1H, 5′-OH); 5.23 (d, J=3.9 Hz, 1H, 3′-OH);6.52 (“t”, J=7.0 Hz, 1H, H-1′); 7.70 (s, 1H, H-6); 8.30 (s, 1H, H-2);8.82 (s, 1H, N═CH). Anal. calculated for C₁₄H₁₈N₅O₃I: C 38.99, H 4.21, N16.24; found: C 39.09, H 4.27, N 16.10.

Example 287-(2-Deoxy-β-D-erythropentofuranosyl)-4-[(dimethylamino)methylidene]amino-5-hexynyl-7H-pyrrolo[2,3-d]pyrimidine(27)

A solution of 5-hexynyl-2′-deoxytubercidin (25) (400 mg, 1.21 mmol) inmethanol (20 ml) is stirred, at 40° C. for 2 h, together withN,N-dimethylformamide dimethyl acetal (2.0 g, 16.8 mmol). After thesolvent has been evaporated off, the residue is purified by flashchromatography (FC) (column: 20×5 cm, CH₂Cl₂/MeOH, 9:1). The main zoneyields a colorless foam (373 mg, 80%). TLC (thin layer chromatography,silica gel, CH₂Cl₂/MeOH, 9:1): R_(f) 0.51. UV (MeOH): max =278 (12100),321 (14300). ¹H-NMR (D₆-DMSO): δ=0.91 (t, J=7.3 Hz, 3H, CH₃); 1.45(sextet, J=7.2 Hz, 2H, CH₂—CH₃); 1.53 (quintet, J=7.3 Hz, 2H,CH₂CH₂—CH₃); 2.18 (m, 1H, H_(α)-2′); 2.47 (m, 3H, H_(β)-2′, C≡C—CH₂,overlapped by DMSO); 3.16, 3.18 (2s, 6H, N(CH₃)₂); 3.56 (m, 2H, H-5′);3.83 (m, 1H, H-4′); 4.35 (m, 1H, H-3′); 5.02 (t, J=5.5 Hz, 1H, 5′-OH);5.26 (d, J=3.9 Hz, 1H, 3′-OH); 6.50 (“t”, J=7.0 Hz, 1H, H-1′); 7.64 (s,1H, H-6); 8.32 (s, 1H, H-2); 8.76 (s, 1H, N═CH).

Example 297-[2-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)-β-D-erythropentofuranosyl]-4-[(dimethylamino)-methylidene]amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidine(28)

4,4′-Dimethoxytriphenylmethyl chloride (256 mg, 0.76 mmol) is added,under an argon atmosphere, to a solution of compound (26) (300 mg, 0.70mmol) in dried pyridine (3 ml). After the mixture has been stirred at50° C. for 2 h, a 5% solution of aqueous NaHCO₃ (15 ml) is added. Theaqueous phase is extracted with CH₂Cl₂ (3 times, 50 ml on eachoccasion). The combined organic phases are dried over Na₂SO₄ and thenevaporated. A colorless foam (360 mg, 70%) is obtained after the residuehas been subjected to flash chromatography (FC) (column: 20×5 cm, B).TLC (silica gel, B): R_(f) 0.60. UV (MeOH) max=236 (38400), 275 (14200),322 (18900).

¹H-NMR (D₆-DMSO): 2.24 (m, 1H, H_(α)-2′); 2.47 (m, 1H, H_(β)-2′,overlapped by DMSO); 3.18 (m, 2H, H-5′), overlapped by NaCH₃); 3.18,3.22 (2s, 6H, N(CH₃)₂); 3.72 (s, 6H, 2 OCH₃); 3.92 (m, 1H, H-4′); 4.37(m, 1H, H-3′); 5.30 (d, J=4.0 Hz, 1H, 3′-OH); 6.54 (“t”, J=6.6 Hz, 1H,H-1′); 6.84 (m, 4H, aromat. H); 7.22-7.38 (m, 9H, aromat. H); 7.56 (s,1H, H-6); 8.31 (s, 1H, H-2); 8.82 (s, 1H, N═CH). Anal. calculated forC₃₅H₃₆N₅O₅I: C 57.30, H 4.95, N 9.55; found: C 57.48, H 5.12, N 9.44.

Example 307-[2-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)-β-D-erythropentofuranosyl]-4-[(dimethylamino)-methylidene]amino-5-hexynyl-7H-pyrrolo[2,3-d]pyrimidine(29)

4,4′-Dimethoxytriphenylmethyl chloride (290 mg, 0.86 mmol) is added,under an argon atmosphere, to a solution of compound (27) (300 mg, 0.78mmol) in dried pyridine (3 ml). After the mixture has been stirred at50° C. for 2 h, a 5% aqueous solution of NaHCO₃ (15 ml) is added. Theaqueous phase is extracted with CH₂Cl₂ (3 times, 50 ml on eachoccasion). The combined organic phases are dried over Na₂SO₄ and thenevaporated. A colorless foam (360 mg, 62%) is obtained following flashchromatography (FC) (column: 20×5 cm, B). TLC (silica gel, B): R_(f)0.60. UV (MeOH) max=276 (17500), 320 (12900). ¹H-NMR (D₆-DMSO): δ=0.91(t, J=7.3 Hz, 3H, CH₃); 1.45 (sextet, J=7.2 Hz, 2H, CH₂—CH₃); 1.53(quintet, J=7.3 Hz, 2H, CH₂—CH₂—CH₃); 2.18 (m, 1H, H_(α)-2′); 2.47 (m,3H, H_(β)-2′, C≡C—CH₂, overlapped by DMSO); 3.16, 3.18 (2s, 6H,N(CH₃)₂); 3.18 (m, 2H, H-5′, overlapped by N(CH₃)₂); 3.71 (s, 6H, 2OCH₃); 3.91 (m, 1H, H-4′); 4.34 (m, 1H, H-3′); 5.28 (d, J=3.9 Hz, 1H,3′-OH); 6.53 (“t”, J=7.0 Hz, 1H, H-1′); 6.82 (m, 4H, aromat. H);7.20-7.36 (m, 9H, aromat. H); 7.56 (s, 1H, H-6); 8.30 (s, 1H, H-2); 8.97(s, 1H, N═CH).

Example 317-[2-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)-β-D-erythropentofuranosyl]-4-[(dimethylamino)-methylidene]amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidine-3′-(2-cyanoethyl)-N,N-diisopropylphosphoramidite(30)

Chloro(2-cyanoethoxy)-N,N-diisopropylaminophosphine (194 mg, 0.82 mmol)is added, under an argon atmosphere, to a stirred solution of the driednucleoside (28) (300 mg, 0.41 mmol) and anhydrousN,N-diisopropylethylamine (212 mg, 1.64 mmol) in dried THF (2 ml). Themixture is stirred for 30 minutes and then filtered. Ethyl acetate (30ml) is added to the filtrate and the whole is extracted twice with anice-cold 10% aqueous solution of Na₂CO₃ (2×10 ml) and 10 ml of water.The organic phases are dried over anhydrous Na₂SO₄ and then evaporated.A colorless foam (222 mg, 60%) is obtained following flashchromatography (FC) (column: 10×3 cm, petroleum ether/acetone). TLC(silica gel, petroleum ether/acetone, 1:1): R_(f) 0.38, 0.45. ³¹P-NMRCDCl₃: 149.0, 149.2.

Example 327-[2-Deoxy-5-O-(4,4′-dimethoxytriphenylmethyl)-β-D-erythropentofuranosyl]-4-[(dimethylamino)-methylidene]amino-5-hexynyl-7H-pyrrolo[2,3-d]pyrimidine-3′-(2-cyanoethyl)-N,N-diisopropylphosphoramidite(31)

Chloro(2-cyanoethoxy)-N,N-diisopropylaminophosphine (194 mg, 0.62 mmol)is added, under an argon atmosphere, to a stirred solution of the driednucleoside 29 (300 mg, 0.44 mmol) and anhydrousN,N-diisopropylethylamine (212 mg, 1.64 mmol) in dried THF (2 ml). Themixture is stirred for 30 minutes and then filtered. Methyl acetate (30ml) is added to the filtrate and the whole is extracted twice with anice-cold 10% aqueous solution of Na₂CO₃ (2×10 ml) and 10 ml of water.The organic phases are dried over anhydrous Na₂SO₄ and then evaporated.A yellow foam (229 mg, 60%) is obtained following flash chromatography(FC) (column: 10×3 cm, petroleum ether/acetone). R_(f) 0.45, 0.53.³¹P-NMR CDCl₃: 149.0, 149.3.

Example 334-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-methylthio-7H-pyrrolo[2,3-d]pyrimidine(32)

The corresponding 5-thiocyanate derivative is formed by proceeding from2′,3′,5′-tri-O-acetyl-7-deazaadenosine and reacting this compound withthiocyanogen chloride in acetic acid. Reduction with 2-mercaptoethanoland subsequent methylation yields the 5-methylthio derivative having anunprotected sugar residue (Watanabe et al., Nucleosides & Nucleotides, 1(2), 191-203, 1982). The methylthio compound (32) is obtained byselective silylation of the 3′,5′-OH groups and subsequent Bartondeoxygenation by way of the corresponding thionoester.

Example 344-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-morpholinomethyl-7H-pyrrolo-[2,3-d]pyrimidine(33)

(33) can be prepared by using the Mannich reaction. The5-morpholinomethyl derivative (Watanabe et al., Nucleosides &Nucleotides, 1(2), 191-203, 1982) is obtained by heating tubercidintogether with paraformaldehyde and morpholine in DMF. The 5-morpholinederivative (33) is obtained in accordance with the silylation anddeoxygenation described in Ex. 33.

Example 354-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-trifluoromethyl-7H-pyrrolo[2,3-d]pyrimidine(34)

Compound 34 is obtained by reacting compound (14) with CF₃Cu inaccordance with the protocol of Nair et al. (J. Am. Chem. Soc., 111,8502-8504, 1989).

Example 364-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-nitro-7H-pyrrolo[2,3-d]pyrimidine(35) and4-amino-7-(2-deoxy-β-D-erythropentofuranosyl)-6-nitro-7H-pyrrolo[2,3-d]-pyrimidine(36)

A mixture composed of 5- and 6-substituted nitro derivatives is obtainedby treating 2′,3′,5′-tri-O-acetyl-7-deazaadenine with HNO₃/H₂SO₄ inmethylene chloride. 5-Nitro derivatives (Watanabe et al., Nucleosides &Nucleotides, 1 (2), 191-203, 1982) result as the main product.Deacylation, 3′,5′-OH silylation and 2′-deoxygenation affords thecompounds (35) and (36).

Example 374-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-6-cyano-7H-pyrrolo[2,3-d]pyrimidine(37)

Oxidation of compound (32) results in the 5-methylsulfone derivative.Treatment with NaCN in DMF yields the 6-cyano derivative (37), aregioisomer of toyocamycin (Watanabe et al., Nucleosides & Nucleotides,1 (2), 191-203, 1982). Silylation and deoxygenation is carried out inaccordance with Ex. 33.

Example 384-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-carboxy-7H-pyrrolo[2,3-d]pyrimidine(38)

Compound (38) is obtained by hydrolyzing compound (37).

Example 394-Amino-5,6-dibromo-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(66)

Method A: NBS (1.42 g, 8.0 mmol, dissolved in 4 ml of dried DMF) isadded, at room temperature, to a solution of 2′-deoxytubercidin (1.0 g,4.0 mmol) and NaOAc (0.78 g, 8.0 mmol) in dried DMF (10 ml). The redsolution is stirred for 10 minutes and then evaporated. Title compound(38) is obtained as colorless crystals (400 mg, 25%) following flashchromatography (column: 20×5 cm, CH₂Cl₂/MeOH, 9:1), evaporation of themore rapidly migrating zone and subsequent recrystallization fromisopropanol. Isolation of the more slowly migrating zone yields5-bromo-2′-deoxytubercidin (130 mg, 10%). Method B: a solution of NBS(1.42 g, 8.0 mmol, dissolved in 4 ml of dried DMF) is added to asolution of 5-bromo-2′-deoxytubercidin (1.3 g, 4.0 mmol) and NaOAc(0.785 g, 8.0 mmol) in dried DMF (10 ml). Purification, see method A.Yield: 490 mg, 30% of 21. Melting point: 181° C. TLC (silica gel,CH₂Cl₂/MeOH, 9:1): R_(f) 0.40. ¹H-NMR (D₆-DMSO): δ=2.12 (m, H_(α)-2′);2.50 (m, H_(β)-2′); 3.51 (m, 2H, H-5′); 3.84 (m, 1H, H-4′); 4.44 (m, 1H,H-3′); 5.20, (br, 2H, 5′-OH, 3′-OH); 6.42 (“t”, J=6.2 Hz, 1H, H-1′);6.93 (br, 2H, NH₂); 8.10 (s, 1H, H-2).

Example 404-Amino-5,6-dichloro-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(39)

The title compound (39) is obtained from the 5-chloronucleoside (1) bychlorinating with N-chlorosuccinimide.

Example 414-Amino-5-iodo-6-chloro-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(40)

The title compound (40) is obtained from the 5-iodonucleoside (14) bychlorinating with N-chlorosuccinimide.

Example 424-Amino-5-iodo-6-bromo-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(41)

The title compound (41) is obtained from the 5-iodonucleoside (14) bybrominating with N-bromosuccinimide.

Example 434-Amino-5-bromo-6-iodo-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(42)

The title compound (42) is obtained from the 5-bromonucleoside (2) byiodinating with N-iodosuccinimide.

Example 444-Amino-5-chloro-6-bromo-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(43)

The title compound (43) is obtained from the 5-chloronucleoside (1) bybrominating with N-bromosuccinimide.

Example 454-Amino-5-chloro-6-iodo-7-(2-deoxy-β-D-erythropentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine(44)

The title compound (44) is obtained from the 5-chloronucleoside (2) byiodinating with N-iodosuccinimide.

Example 465-Chloro-7-[2-deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-2-[(formyl)amino]-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine(45)

N-Chlorosuccinimide (87 mg, 0.67 mmol) is added to a solution ofcompound (18) (300 mg, 0.67 mmol in 5 ml of DMF). After it has beenstirred (at room temperature for 20 h), the solution is added to amixture of CH₂Cl₂/5% aq. NaHCO₃ (50 ml, 9:1). The organic layer isseparated off, washed with water, dried over Na₂SO₄, filtered andevaporated. The evaporated residue is dissolved in CH₂Cl₂ and thissolution is chromatographed on silica gel (column: 5×20 cm,CH₂Cl₂/acetone, 95:5). The main zone is concentrated, and n-hexane isadded to the residue. The precipitated colorless crystals (230 mg, 71%)are isolated. Melting point: 119-120° C. ¹H-NMR ([D₆] DMSO): δ=1.09 (d,J=7.0, CH₃), 1.15 (d, J=6.9, CH₃), 2.45, 2.60, 2.63, 2.89 (4m, CH andH_(α,β)-C(2′)), 4.06 (s, OCH₃), 4.18 (m, H—C(5′)), 4.28 (m, H—C(4′)),5.35 (m, H—C(3′)), 6.47 (m, H—C(1′)), 7.58 (s, H—C(6)), 9.45 (d, J=8.9,NH), 10.84 (d, J=9.6, HCO). Anal. calculated for C₂₁H₂₇CIN₄O₇ (482.9): C52.23, H 5.64, N 11.60; found: C 52.51, H 5.69, N 11.65.

Example 477-[2-Deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-2-[(formyl)amino]-5-iodo-4-methoxy-7H-pyrrolo-[2,3-d]pyrimidine(46)

Compound (46) is prepared as described for compound (45) by proceedingfrom compound (18) (500 mg, 1.11 mmol) and N-iodosuccinimide (264 mg,1.16 mmol). The duration of the reaction is 23 h. Colorless crystals(590 mg, 92%) are obtained from cyclohexane. ¹H-NMR ([D₆] DMSO): δ=1.9(d, J=7.0, CH₃), 1.15 (d, J=7.0, CH₃), 2.46, 2.60, 2.89 (3 m, CH andH_(α,β)-C(2′)), 4.06 (s, OCH₃), 4.17 (m, H—C(5′)), 4.27 (m, H—C(4′)),5.35 (m, H—C(3′)), 6.46 (m, H—C(1′)), 7.62 (s, H—C(6)), 9.55 (d, J=9.7,NH), 10.81 (d, J=9.9 HCO). Anal. calculated for C₂₁H₂₇JN₄O₇ (574.4): C43.91, H 4.74, N 9.75; found: C 43.98, H 4.75, N 9.82.

Example 486-Chloro-7-[2-deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-2-[(formyl)amino]-5-iodo-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine(47)

The title compound (47) is obtained from the 5-iodonucleoside (46) bychlorinating with N-chlorosuccinimide.

Example 495-Bromo-6-chloro-7-[2-deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-2-((formyl)amino-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine(48)

The title compound (48) is obtained from the 5-bromonucleoside (2) bychlorinating with N-chlorosuccinimide.

Example 507-[2-Deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-5,6-dichloro-2-[(formyl)amino]-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine (49)

N-Chlorosuccinimide (117 mg, 0.9 mmol) is added to a solution ofcompound (45) (200 mg, 0.4 mmol in 5 ml of DMF). After it had beenstirred (at room temperature for 16 h), the solution is added to amixture of CH₂Cl₂/5% aq. NaHCO₃ (50 ml, 9:1). The organic phase isseparated off, washed with water, dried over Na₂SO₄, filtered andevaporated. The evaporated residue is dissolved in CH₂Cl₂ and thissolution is chromatographed on silica gel (column: 5×20 cm,CH₂Cl₂/acetone, 95:5). The main zone is separated off. Colorlesscrystals (149 mg, 72%) are obtained after evaporating off the solventand crystallizing from cyclohexane. Melting point: 127-128° C. ¹H-NMR([D₆] DMSO): δ=1.01 (dd, CH₃), 1.14 (d, J=7.0, CH₃), 2.44, 2.47, 2.60,3.53 (4 m, CH and H_(αβ)-C(2′)), 4.05 (s, OCH₃), 4.17 (m, H—C(5′)), 4.30(m, H—C(4′)), 5.56 (m, H—C(3′)), 6.41 (m, H—C(1′)), 9.44 (d, J=9.1, NH),10.94 (d, J=9.8, HCO). Anal. calculated for C₂₁H₂₆Cl₂N₄O₇ (517.4): C48.75, H 5.07, N 10.83; found: C 49.04, H 5.09, N 10.66.

Example 515-Chloro-6-iodo-7-[2-deoxy-3,5-di-O-(2-methylpropionyl)-β-D-erythropentofuranosyl]-2-[(formyl)amino]-4-methoxy-7H-pyrrolo[2,3-d]pyrimidine (50)

The title compound (50) is obtained from the 5-chloronucleoside (45) byiodinating with N-iodosuccinimide.

Example 522-Amino-5,6-dichloro-7-(2-deoxy-β-D-erythropentofuranosyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(51)

A suspension of compound (49) (200 mg, 0.4 mmol) in 2N aq. NaOH (8 ml)is boiled under reflux for 3 h. After the solution has been neutralizedwith conc. AcOH, the reaction product is filtered, washed with water anddried. Colorless crystals (128 mg, 96%) are obtained after crystallizingfrom CH₃CN. ¹H-NMR ([D₆] DMSO): δ=2.22 (m, H_(α)—C(2′)), 2.92 (m,H_(β)—C(2′)), 3.52 (m, H—C(5′)), 3.72 (m, H—C(4′)), 4.33 (m, H—C(3′)),4.81 (t, 5′-OH), 5.22 (d, 3′-OH), 6.35 (dd, H—C(1′)), 6.46 (br., NH₂),10.75 (s, NH).

Example 532-Amino-5-iodo-6-chloro-7-(2-deoxy-β-D-erythropentofuranosyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(52)

The title compound (52) is prepared as described under Example 52 byproceeding from compound (47).

Example 542-Amino-5-bromo-6-chloro-7-(2-deoxy-β-D-erythropentofuranosyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(53)

The title compound (53) is prepared as described under Example 52 byproceeding from compound (48).

Example 552-Amino-5-chloro-6-iodo-7-(2-deoxy-β-D-erythropentofuranosyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(54)

The title compound (54) is prepared as described under Example 52 byproceeding from compound (50).

Example 562-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(55)

The title compound (55) is prepared by the method described in Winkeleret al. (Liebigs Ann. Chem. 1984, 708).

Example 572-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-6-methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(56)

The title compound (56) is prepared in analogy with Example 55.

Example 582-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5,6-dimethyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(57)

The title compound (57) is prepared in analogy with Example 55

Example 592-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-iodo-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(58)

The title compound (58) is prepared in analogy with Example 51 byproceeding from compound (46) (200 mg, 0.35 mmol). Colorless crystals(126 mg, 92%) are obtained from MeCN. Melting point: 218-220° C. UV(MeOH): λ_(max) 266 (12,000), 285 (sh, 8400). ¹H-NMR ([D₆] DMSO): δ=2.08(m, H_(α)—C(2′)), 2.30 (m, H_(β)—C(2′)), 3.48 (m, H—C(5′)), 3.74 (m,H—C(4′)), 4.26 (m, H—C(3′)), 4.89 (t, 5′-OH), 5.18 (d, 3′-OH), 6.25 (dd,H—C(1′)), 6.34 (br., NH₂), 7.09 (s, H—C(6)), 10.51 (br., NH). Anal.calculated for C₁₁H₁₃IN₄O₄ (392.2): C 33.69, H 3.34, N 14.29; found: C33.78, H 3.42, N 14.29.

Example 607-(2-Deoxy-β-D-erythropentofuranosyl)-5-iodo-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(59)

After it has been dried three times by evaporating off pyridine,compound (58) (300 mg, 0.76 mmol; dissolved in 4 ml of dried pyridine)is treated with 0.48 ml (3.78 mmol) of trimethylchlorosilane. Thesolution is stirred for 15 min. After 0.62 ml (3.78 mmol) of isobutyricanhydride has been added, the solution is left to stand at roomtemperature for 3 h. After the reaction mixture has been cooled down inan ice bath, 1 ml of water is added. After 5 min, 1 ml of a 25% strengthaqueous solution of ammonia is added and the mixture is stirred for 15min. The solution is then evaporated almost to dryness. Colorlesscrystals (312 mg, 89%) are obtained following crystallization fromwater. ¹H-NMR ([D₆]) DMSO): δ=1.10 (2 CH₃), 2.12 (m, H_(β)—C(2′)), 2.37(m, H_(α)—C(2′)), 2.73 (m, CH), 3.50 (m, H—C(5′)), 3.78 (m, H—C(4′)),4.30 (m, H—C(3′)), 4.89 (t, 5′-OH), 5.20 (d, 3′-OH), 6.35 (dd, H—C(I′)),7.43 (s, H—C(6)), 11.49, 11.76 (2s, 2 NH). Anal. calculated forC₁₅H₁₉IN₄O₅ (462.2): C 38.98, H 4.14, N 12.12; found: C 39.11, H 4.37, N11.96.

Example 617-(2-Deoxy-β-D-erythropentofuranosyl)-5-methyl-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(60)

After having been dried three times by evaporating off pyridine,compound (55) (500 mg, 1.78 mmol; dissolved in 9 ml of dried pyridine)is treated with 1.2 ml (9 mmol) of trimethylchlorosilane. The solutionis stirred for 15 min. After 1.2 ml (9 mmol) of isobutyric anhydride hasbeen added, the solution is left to stand at room temperature for 3 h.After the reaction mixture has been cooled down in an ice bath, 1.8 mlof water are added. After 5 min, 1.8 ml of a 25% strength aqueoussolution of ammonia are added and the reaction is continued for afurther 15 min. The mixture is then evaporated almost to dryness and theresidue is taken up in 10 ml of water, whereupon colorless crystals (555mg, 89%) crystallize out very rapidly. Thin layer chromatography (silicagel, CH₂Cl₂/MeOH, 9:1): R_(f)=0.7. ¹H-NMR [D₆-DMSO] 1.10 (d, J=6.5 Hz,2CH₃—C), 2.11, 2.28 (2m, 2H—C(2′)), 2.23 (s, CH₃), 2.73 (q, J=6.6 Hz,CH), 3.48 (m, 2H—C(5′)), 3.75 (m, H—C(4′)), 4.29 (m, H—C(3′)), 4.85 (m,OH—C(5′)), 5.20 (m, OH—C83′)), 6.36 (t, J=6.7 Hz, H—C(1′)), 6.94 (s,H—C(6)), 11.42, 11.67 (2s, 2NH). Anal. calculated for C₁₆H₂₂N₄O₅(350.37): C 54.84, H 6.33, N 15.99; found: C 54.76, H 6.46, N 16.01.

Example 627-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-β-D-erythropentofuranosyl]-5-iodo-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(61)

Compound (59) is repeatedly dried by evaporating off anhydrous pyridine.400 mg (0.87 mmol) of compound (59) which has been dried in this way aredissolved in 5 ml of anhydrous pyridine. After adding4,4′-dimethoxytrityl chloride (328 mg, 0.95 mmol) at room temperature,the reaction mixture is stirred overnight. MeOH (3 ml) and 5% aq. NaHCO₃(30 ml) are then added. The aqueous phase is extracted 3 times withCH₂Cl₂. The organic phase is dried (Na₂SO₄) and evaporated, and theresidue is subjected to flash chromatography (column: 10×5 cm, solventCH₂Cl₂/acetone, 9:1). Isolating the material from the main zone affordsthe colorless, amorphous title compound (61). (600 mg, 91%). ¹H-NMR([D₆] DMSO: δ=1.13 (m, 4 CH₃), 2.24 (m, H—C(2′)), 2.77 (m, CH), 3.12 (m,H—C(5′)), 3.75 (s, 2 CH₃O), 3.93 (m, H—C(4′)), 4.35 (m, H—C(3′)), 5.30(d, 3′-OH), 6.39 (dd, H—C(1′)), 6.86-7.39 (m, aromatic H +H—C(6)),11.54, 11.82 (2s, 2 NH). Anal. calculated for C₃₆H₃₇JN₄O₇ (764.6): C56.55, H 4.88, N 7.33: found C 56.42, H 4.82, N 7.30.

Example 637-[2-Deoxy-5-O-(4,4-dimethoxytrityl)-β-D-erythropentofuranosyl]-5-methyl-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(62)

(MeO)₂TrCl (448 mg, 1.3 mmol) is added to compound (60) (390 mg, 1.1mmol; dried by evaporating from dry pyridine, suspended in 8 ml of driedpyridine), and the reaction mixture is stirred at room temperature for 4h. After MeOH (5 ml) has been added, the reaction mixture is treatedwith 5% aq. NaHCO₃ (80 ml). After extracting with CH₂Cl₂ (3×50 ml), theorganic phases are combined, dried (anhydrous NaSO₄) and evaporated. Theremaining residue is dissolved in CH₂Cl₂ and this solution is subjectedto flash chromatography. (Silica gel column: 4×8 cm, solventCH₂Cl₂/MeOH, 95:5 containing traces of Et₃N). The main zone is isolatedand title compound (62) is obtained as a colorless powder (654 mg, 90%).Thin layer chroamtography (silica gel, CH₂Cl₂/MeOH, 95:5): R_(f)=0.3.¹H-NMR (d₆-DMSO): 1.10 (d, J=6.7 Hz, 2CH₃—C); 2.16 (s, CH₃); 2.20, 2.40(2m, 2H—C(2′)); 2.74 (q, J=6.8 Hz, CH); 3.12 (m, H—C(5′)); 3.72 (s,2CH₃O); 3.89 (m, H—C(4′)); 4.34 (m, H—C(3′)); 5.30 (d, J=3.7 Hz,OH—C(3′)); 6.38 (t, J=6.7 Hz, H—C(1′)); 6.7-7.4 (m, aromatic H andHC(6)); 11.46, 11.71 (2s, 2NH). Anal. calculated for C₃₇H₄₀N₄O₅(652.72): C 68.08, H 6.18, N 8.58; found: C 68.25, H 6.29, 8.50.

Example 647-2-[Deoxy-5-O-(4,4-dimethoxytrityl)-β-D-erythropentofuranosyl]-5-iodo-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one-3′-triethylammoniumPhosphonate (63)

1,2,4-Triazole (480 mg, 6.8 mmol) is added to a solution of PCl₃ (180μl, 2 mmol) and N-methylmorpholine (2.2 ml) in CH₂Cl₂ (12 ml). Thesolution is stirred for 30 min and then slowly cooled down to 0° C. 306mg (0.4 mmol) of compound (61) (dissolved in 12 ml of CH₂Cl₂) are slowlyadded and the mixture is stirred at 0° C. for 30 min. After that, it isadded to 1 M (Et₃NH)HCO₃ (TBC, pH 8.0, 25 ml), and the whole is shakenand the phases separated. The aqueous phase is extracted with CH₂Cl₂(3×30 ml). The combined organic extracts are dried (Na₂SO₄) andevaporated. Following chromatography (columns: 10×5 cm, solventCH₂Cl₂/Et₃N, 98:2, then CH₂Cl₂/MeOH/Et₃N (88:10:2), title compound (63)(320 mg, 86%) is obtained as a colorless foam after extracting with 0.1M TBC (8×20 ml), drying with NCl₂SO₄ and evaporating. ¹H-NMR ([D₆]DMSO): δ=1.15 (m, 5 CH₃), 2.36-2.37 (m, H—C(2′)), 2.76 (m, CH), 2.98 (m,3 CH₂), 3.20 (m, H—C(5′)), 3.75 (s, 2 MeO), 4.11 (m, H—C(4′)), 4.80 (m,H—C(3′)), 6.44 (dd, H—C(1′)), 6.09 (s, pH), 6.87-7.39 (m, aromaticH+H—C(6)), 11.79 (br., 2 NH). ³¹p-NMR ([D₆]DMSO): 1.05 (′J(P,H)=587,³J(p,H—C(3′))=8.3 Hz.

Example 657-[2-Deoxy-5-O-(4,4-dimethoxytrityl)-β-D-erythropentofuranosyl]-5-methyl-2-isobutyrylamino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one,3′-triethylammonium Phosphonate (64)

1,2,4-Triazole (523 mg, 7.3 mmol) is added to a solution of PCl₃ (200μl, 2.26 mmol) and N-methylmorpholine (2.5 ml) in CH₂Cl₂ (14 ml). Thesolution is stirred for 30 min and then slowly cooled down to 0° C. 300mg (0.46 mmol) of compound (62) (dissolved in 14 ml of CH₂Cl₂) areslowly added, and the mixture is stirred at room temperature for 30 min.After that; it is added to 1 M (Et₃NH)HCO₃ (30 ml), and the whole isshaken and the phases separated. The aqueous phase is extracted withCH₂Cl₂ (3×40 ml). The combined organic extracts are dried with anhydrousNa₂SO₄ and concentrated. The concentrated residue is subjected to flashchromatography (silica gel, 3×7 cm column, CH₂Cl₂/MeOH/Et₃N, 88:10:2).The main zone fractions are collected and evaporated; the residue isdissolved in CH₂Cl₂ and this solution is washed with 0.1 M (Et₃NH)HCO₃(5×15 ml). The organic phase is dried with anhydrous Na₂SO₄ andevaporated. The title compound (64) is obtained as a colorless foam (270mg, 72%). Thin layer chromatography (silica gel, CH₂Cl₂/MeOH/Et₃N,88:10:2): R_(f)=0.5. ¹H-NMR (D₆-DMSO): 1.16 (m, 5CH₃); 2.19 (s, CH₃);2.30, (m, H—C(2′)); 2.74 (q, J=6.3 Hz, CH); 3.00 (q, J 6.4 Hz, 3CH₂);3.13, 3.18 (2m, 2H—C(5′)); 3.75 (s, CH₃O); 4.01 (m, H—C(4′)); 4.77 (m,H—C(3′)); 6.43 (d, J (P,H)=346 Hz, PR); 6.45 (t, J=6.7 Hz H—C(1′));6.8-7.4 (m, aromatic H and H—C(6)); 11.67, 11.69 (2s, 2NH). ³¹P-NMR(d₆-DMSO): 0.94 (¹J (P,H)=354 Hz, ³J (P,H—C(3′))=8.1 Hz). Anal.calculated for C₄₃H₅₆N₅O₉P (817.89): C 63.14, H 6.90, N 8.56; found: C63.06, H 6.88, N 8.51.

Example 662-Amino-7-(2-deoxy-β-D-erythropentofuranosyl)-5-(1-hexynyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one(65)

Copper iodide (38.1 mg, 0.2 mmol), triethylamine (2.8 ml, 2 mmol),tetrakis(triphenylphsophine)palladium(0) (40.5 mg, 0.1 mmol) and1-hexyne (492 mg, 6 mmol) are added to an argon-flushed solution ofcompound (58) (390 mg, 1 mmol in 5 ml of dried DMF), and the solution isstirred at room temperature for 24 h. It is then evaporated and theresidue is loaded onto a silica gel column (5×25 cm). Title compound(65) is obtained following stepwise elution with 5%, 10% and 20% MeOH inCH₂Cl₂. Recrystallization from MeCN affords a colorless solid (120 mg,35%). ¹H-NMR ([D₆] DMSO): δ=0.94 (m, CH₃), 1.49, 2.38 (m, CH₂), 2.0 (M,H_(α)—C(2′)), 2.35 (m, H_(β)—C(2′)), 3.50 (m, H₂—C(5′)), 3.76 (m,H—C(4′)), 4. (m, H—C(3′)), 4.88 (t, 5′-OH), 5.18 (d, J=3.5, 3′-OH), 6.27(m, H—C(1′)+NH₂), 7.13 (s, H—C(6)), 10.34 (br., NH). MS: m/e 346 (M⁺).

Example 67 Solid Phase Synthesis of the Oligodeoxyribonucleotides by thePhosphonate Method

The oligodeoxyribonucleotide syntheses were carried out on a 1 μmolscale on a solid phase (CPG: ®Controlled Pore Glass) in an automatedmodel 380 B DNA synthesizer (Applied Biosystems, Weiterstadt) by meansof the phosphonate technique, with the DNA fragment being synthesized inthe 3′-5′ direction. The reaction cycle (detritylation, coupling,capping and oxidation) followed a program which was developed forphosphonate chemistry [H. Köster, K. Kulikowsky, T. Liese, W. Heikens,V. Kohli, Tetrahedron 1981, 37, 363]. The base-protectedoligonucleotide, whose 5′-hydroxyl group was also protected by Dmt, wascleaved from the support within 30 min using 25% aqueous ammonia. Theprotective groups on the heterocycles were removed at 60° C. in the samemedium within 48 h. While adding a drop of triethylamine (to avoidpremature elimination of the 5′-OH protective group) the samples wereconcentrated down to about 200 μl in a Speed-Vac ®concentrator. In thisstate, they can be kept at −25° C. for some months.

Example 68 Solid Phase Synthesis of the Oligodeoxyribonucleotides by thePhosphoramidite Method.

The oligodeoxyribonucleotide syntheses were carried out on a 1 μmolscale in an automated model 380 B DNA synthesizer (Applied Biosystems,Weiterstadt) by means of the solid phase phosphoramidite technique using®CPG (controlled pore glass) or ®Fractosil to which the first nucleosideunit is bonded via its 3′ end. The following steps were carried out:

1. Washing with abs. acetonitrile, 2. treating with 3% trichloroaceticacid in dichloro- methane, 3. washing with abs. acetonitrile, 4.condensing with 10 μmol of 5′-O-dimethoxytrityl-nucleoside-3′-β-cyanoethyl phosphite-diisopropyl- amidite and 50 μmol oftetrazole in 0.3 ml of abs. acetonitrile, 5. washing with acetonitrile,6. capping with 20% acetic anhydride in THF containing 40% lutidine and10% dimethylaminopyridine, 7. washing with acetonitrile, 8. oxidizingwith iodine (1.3 g in THF/water/pyridine; 70:20:5 = v:v:v).

Steps 1 to 8, termed a DNA reaction cycle below, were repeated in orderto assemble the oligonucleotide in accordance with the sequence to besynthesized, with the 5′-O-dimethoxytrityl(nucleotidebase)-3′-β-cyanoethyl phosphite-diisopropylamidite, which in each casecorresponded to the sequence, being employed in step 4. Once thesynthesis is complete, working-up is carried out as described in Example67.

Example 69 HPLC Purification of the Trityl-protected and DeprotectedOligonucleotides

In the first purification step, the DMT-protected oligomers werepurified by HPLC on RP-18 silica gel (eluent system I, see below),evaporated to dryness, reevaporated several times with dry methanol, andsubsequently detritylated, while cooling with ice, by 10-minute exposureto 80% strength acetic acid. After that, the acid was neutralizeddropwise with triethylamine (1-2 ml) at 0° C., concentrated almost todryness and then reevaporated twice with absolute methanol. After theresidue had been taken up in 500 μl of double-distilled water, thecompletely deprotected oligonucleotides were once again purified by HPLCon RP-18 silica gel (eluent system II, see below). The combined mainzones were evaporated and the residue was dissolved in 500 μl ofdouble-distilled water, and this solution was desalted through a shortRP-18 column (eluent system III, see below). After having beenlyophilized in a Speed-Vac concentrator, the oligonucleotides were takenup in 100 μl of double-distilled water and this solution was stored at−25° C.

The following HPLC eluents were used:

A: 0.1 N triethylammonium acetate, pH 7.0/5% strength acetonitrile B:double-distilled water C: acetonitrile D: methanol/water, 3:2

The following gradient systems, composed of the above eluents, wereemployed:

-   -   I: 3 min. 15% C in A, 7 min. 15-40% C in A, 5 min. 40% C in A, 5        min. 40-15% C in A    -   II: 20 min. 0-20% C in A, 5 min. 20% C in A    -   III: 100% A    -   IV: 30 min. B, 10 min. D    -   V: 12 min. 100% A, 8 min. 0-40% C in A, 5 min. 40-0% C in A

The oligomers were observed to have the following retention times:

HPLC retention times of the synthesized oligonucleotides: Retentiontimes [min] Oligomer (5′ → 3′ direction) with trityl without trityl d(A₁₂) 11.6 15.5 (SEQ ID NO.: 29) d (T₁₂) 11.5 13.7 (SEQ ID NO.: 30) d(c⁷A₁₁A) 12.3 15.3 d (Br⁷C⁷A₁₁A) 12.7 19.1 d (Me⁷c⁷A₁₁A) 12.2 18.1 d(A-T)₆ 13.5 20.1 (SEQ ID NO.: 31) d (c⁷A-T)₆ 13.6 20.5 d (Cl⁷c⁷A-T)₆12.8 19.9 d (Br⁷c⁷A-T)₆ 12.3 17.8 d (Me⁷c⁷A-T)₆ 12.6 17.9

Example 70 Characterization of the Oligodeoxyribonucleotides by Means ofEnzymic Hydrolysis

The oligonucleotides (0.5 A₂₆₀ units in each case) are dissolved in 0.1M tris-HCl buffer (pH=8.3, 200 μl) and incubated with snake venomphosphodiesterase (3 μg) at 37° C. for 45 min. Alkaline phosphatase (3μg) is then added and the temperature is maintained at 37° C. for afurther 30 min. The resulting nucleoside mixture is analyzed by means ofUV spectrophotometry using reversed-phase HPLC (eluent system V). Thenucleoside composition of the corresponding oligonucleotide can bequantified on the basis of the peak areas and the extinctioncoefficients of the nucleosides at 260 nm (dA: 15400, dC: 7300, dG:11700, dT: 8800, Brdc⁷A: 5300, Medc⁷A: 4900, Cldc⁷A: 6300).

Example 71 Determination of Cleavage Hypochromicity by Means of theEnzymic Hydrolysis of the Oligonucleotides

0.2 A₂₆₀ units of the oligonucleotide are hydrolyzed with snake venomphosphodiesterase in 0.1 M tris-HCl buffer (pH=8.3, 200 μl). From the UVabsorption at 260 nm before and after cleaving, the hypochromicity in %can be calculated, while taking into account the enzyme absorption, inaccordance with the following equation:H _(enzyme)=[(ε_(monomer)−ε_(polymer))×(ε_(monomer))⁻¹]×100%

Example 72 UV-Spectroscopic and CD-Spectroscopic Determination of theT_(m) Values and Calculation of the Thermodynamic Data

The T_(m) values of the oligomers were determined using a Cary 1 UV-Visspectrophotometer (Varian, Melbourne, Australia). The temperature wasvaried linearly by 0.5° C. or 1.0° C. per minute. In order toinvestigate the melting temperature, oligomer concentrations of between0.2 and 0.8 A₂₆₀ units in 1 ml of 60 mM sodium cacodylate buffer (pH7.5, 1 M NaCl, 100 mM MgCl₂) were used. In the experiments on thenon-self-complementary oligonucleotides, the single-strand concentrationwas 0.2-0.6 OD. The melting hypochromicity in % is obtained from thedifference in absorption before and after melting in accordance with thefollowing equation:H _(melt.)=[(A _(e) −A _(t))A _(e) ⁻¹]×100The melting curves were analyzed using a program based on a two-statemodel (“stacked/unstacked”) in accordance with the equation:ln K=ln [(E ^(S) −E)/(E ^(U) −E)]=S/R−H/RTwhere E=absorption at the corresponding wavelength, S=stacked andU=unstacked. The temperature-dependent CD spectra were plotted in awavelength range of 200-350 nm using a Jasco 600 spectropolarimeter witha thermostated quartz cuvette. The temperature was increased inintervals of 5-10° C. in a range of from 5-80° C. at concentrations offrom 3 to 15 μM in 60 mM Na cacodylate buffer and at 0.1 M, 1 and 4 MNaCl.

Example 73

T_(m) values of the oligonucleotides^(a) Oligomer T_(m) [° C.] d(A₁₂)(SEQ ID NO.: 29) × 44^(b) d(T₁₂) (SEQ ID NO.: 30) d(c⁷A₁₁A) × 30^(b)d(T₁₂) (SEQ ID NO.: 30) d(Br⁷c⁷A₁₁A) × 53^(b) d(T₁₂) (SEQ ID NO.: 30)d(Me⁷c⁷A₁₁A) × 48^(b) d(T₁₂) (SEQ ID NO.: 30) [d(A—T)₆]₂ 33^(c) (SEQ IDNO.: 31) [d(c⁷A—T)₆]₂ 36^(c) [d(Br⁷c⁷A—T)₆]₂ 55^(c) [d(Cl⁷c⁷A—T)₆]₂59^(c) [d(Me⁷c⁷A—T)₆]₂ 41^(c) [d(hexynyl⁷c⁷A—T)₆]₂ 50^(d) [d(I⁷c⁷A—T)₆]₂60^(d) [d(G—C)₄]₂ 60^(d) (SEQ ID NO.: 32) [d(c⁷G—C)₄]₂ 53^(d)[d(Me⁷c⁷G—C)₄]₂ 58^(d) [d(I⁷c⁷G—C)₄]₂ 70^(d) ^(a)determined in 1 M NaCicontaining 60 mM Na cacodylate, 100 mM MgCl₂, pH 7.1 ^(b)oligomerconcentration: 7.5 μM single-stranded ^(c)oligomer concentration: 15 μMsingle-stranded ^(d)oligomer concentration: 10 μM single-stranded

Example 74 Phosphodiester Hydrolysis of Self-complementaryOligonucleotides with EcoRI Endodeoxyribonuclease

0.5 A₂₆₀ units of the appropriate oligonucleotide are dissolved in 100μl of buffer (composed of 50 μM tris-HCl buffer, pH 7.5, 100 mM NaCl, 10mM magnesium chloride and 1 mM dithioerythritol), and EcoRIendodeoxyribonuclease (high concentration, 5 μl ≡250 units) is added tothis solution. The mixture was then incubated at 37° C. and samples ofin each case 10 μl in volume were removed at 30 min intervals andanalyzed by reverse-phase HPLC (eluent system II).

Example 75 Testing for Nuclease Stability

10 mmol of the oligonucleotide to be investigated are dissolved in 450μl of 20% strength fetal calf serum in RPMI medium and 50 μl ofdouble-distilled water, and this solution is incubated at 37° C. 10 μlsamples for gel electrophoresis or 20 μl samples for HPLC are thenremoved immediately and after 1, 2, 4, 7 and 24 hours; the reaction isterminated by adding 5 μl of 10 μl of formamide and heating at 95° C.for 5 minutes. For the gel electrophoresis, the samples are loaded ontoa 15% polyacrylamide gel (2% BIS), which is developed at about 3000 volthours. The bands are visualized by silver staining. For the HPLCanalysis, the samples are injected onto a Gen-Pak fax HPLC column(Waters/Millipore) and chromatographed at 1 ml/min using from 5 to 50%buffer A in B (buffer A: 10 mM sodium dihydrogen phosphate, 0.1 M NaClin acetonitrile/water, 1:4 (v:v) pH 6.8; buffer B: as A, but containing1.5 M NaCl).

Example 76 Testing for Antiviral Activity

The antiviral activity of the test substances against various herpesviruses which are pathogenic to humans is examined in a cell culturetest system. For the experiment, monkey kidney cells (Vero, 2×10⁵/ml) inserum-containing Dulbecco's MEM (5% fetal calf serum, FCS) are sown in96-well microtiter plates, which are incubated at 37° C. for 24 h in 5%CO₂. The serum-containing medium is then sucked off and the cells arerinsed twice with serum-free Dulbecco's MEM (-FCS). The test substancesare prediluted in H₂O to a concentration of 600 μM and stored at −18° C.Further dilution steps in Dulbecco's minimal essential medium (MEM) arecarried out for the test. 100 μl of each of the individual testsubstance dilutions are added, together with 100 μl of serum-freeDulbecco's MEM (-FCS), to the rinsed cells. After having been incubatedat 37° C. for 3 h in 5% CO₂, the cells are infected with herpes simplexvirus type 1 (ATCC VR733, HSV-1 F strain) or with herpes simplex virustype 2 (ATCC VR734, HSV-2 G strain) in concentrations at which the celllawn is completely destroyed within 3 days. In the case of HSV-1, theinfection intensity is 500 plaque-forming units (PFU) per well, and inthe case of HSV-2 it is 350 PFU/well. The experimental mixtures thencontain test substance at concentrations of from 80 μM to 0.04 μM inMEM, supplemented with 100 U/ml penicillin G and 100 mg/l streptomycin.All the experiments are carried out as duplicate determinations apartfrom the controls, which are carried out eight times per plate. Theexperimental mixtures are incubated at 37° C. for 17 h and in 5% CO₂.The cytotoxicity of the test substances is determined after a totalincubation time of 20 h by microscopic assessment of the cell cultures.The highest preparation concentration which still does not cause anymicroscopically recognizable cell damage under the specifiedexperimental conditions is designated the maximum tolerated dose (MTD).FCS is then added to a final concentration of 4% and the plates areincubated at 37° C. in 5% CO₂ for a further 55 h. The untreatedinfection controls then exhibit a fully developed cytopathic effect(CPE). After the cell cultures have been assessed microscopically, theyare stained with neutral red using Finter's (1966) vital stainingmethod. The antiviral activity of a test substance is defined as theminimum inhibitory concentration (MIC) which is required in order toprotect 30-60% of the cells from the cytopathic effect of the virus.

Abbreviations: Bz benzoyl br. broad CD circular dichroism d doublet TLCthin layer chromatography dG 2′-deoxyguanosine dA 2′-deoxyadenosine dC2′-deoxycytidine dT 2′-deoxythymidine (D₆)DMSO dimethyl sulfoxide,deuterated 6 times DMF dimethylformamide DNA deoxyribonucleic acid Dmt4,4′-dimethoxytrityl (4,4′-dimethoxy- triphenylmethyl) EDTAethylenediamine tetraacetate EtOAc ethyl acetate Et₃N triethylamine FCflash chromatography G free enthalpy h hour H enthalpy of duplexformation HPLC high pressure liquid chromatography Hyp. hypochromicityibu isobuturyl J coupling constant K_(m) Michaelis-Menten constant NMRnuclear magnetic resonance PAGE polyacrylainide gel electrophoresis PCRpolymerase chain reaction ppm parts per million 2-PrOH isopropanol R_(f)retention in TLC relative to the eluent front RNA ribonucleic acid RPreverse phase s singlet S entropy of duplex formation M.p. melting pointSVPD snake venom phosphodiesterase t triplet TBC triethylammoniumbicarbonate T_(m) oligomer melting temperature UV ultraviolet v_(max)maximum reaction velocity c⁷A 7-deazaadenosine Br⁷c⁷A7-bromo-7-deazaadenosine Cl⁷c⁷A 7-chloro-7-deazaadenosine I⁷c⁷A7-iodo-7-deazaadenosine Me⁷c⁷A 7-methyl-7-deazaadenosine c⁷G7-deazaguanosine I⁷c⁷G 7-iodo-7-deazaguanosine Me⁷c⁷7-methyl-7-deazaguanosine λ wavelength ε molar extinction coefficient

1. A nucleotide monomer of the formula V

in which V is oxy, sulfanediyl or imino; Y^(b) is oxy, sulfanediyl ormethylene; a is oxy, sulfanediyl or methylene; R^(2b) is hydrogen, OR¹²,C₁-C₆-alkenyloxy, in particular allyloxy, halogen, azido or NR¹⁰R¹¹; R¹is a protective group which is customary in nucleotide chemistry; R^(1b)is a linker, optionally linked to a solid support, or is a radical ofthe formula IIIc or IIId

in which U is (C₁-C₁₈)-alkoxy, (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl,(C₆-C₁₄)-aryl-(C₁-C₈)-alkyl, O—R⁷, S—R⁷ or a radical of the formula IV(OCH₂CH₂)_(p)O(CH₂)_(q)CH₂R⁵  (IV) in which R⁵ is H; Q is a radical—NR⁸R⁹, R⁷ is —(CH₂)₂—CN; R⁸ and R⁹ are identical or different and areC₁-C₆-alkyl, or, together with the nitrogen atom carrying them, are a5-9-membered heterocyclic ring which can additionally contain a furtherO, S or N heteroatom; E′ and F′ are, independently of each other, H,OR¹² or NR¹⁰R¹¹, R¹⁰ and R¹¹ are identical or different and are hydrogenor an amino protective group which is customary in nucleotide chemistry,or R¹⁰ and R¹¹ together form an amino protective group which iscustomary in nucleotide chemistry, R¹² is hydrogen or a hydroxylprotective group which is customary in nucleotide chemistry; R¹⁵ and R¹⁶are, independently of each other, (1) hydrogen, (2) halogen, (3)(C₁-C₁₀)-alkyl, (4) (C₂-C₁₀)-alkenyl, (5) (C₂-C₁₀)-alkynyl, (6) NO₂, (7)NH₂, (8) cyano, (9) —S—(C₁-C₆)-alkyl, (10) (C₁-C₆)-alkoxy, (11)(C₆-C₂₀)-aryloxy, (12) SiH₃

(14) a radical as described under (3), (4) or (5) which is substitutedby one or more radicals selected from the group consisting of SH,S—(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —NR(c)R(d), —CO—R(b),—NH—CO—NR(c)R(d), —NR(c)R(g), —NR(e)R(f), and —NR(e)R(g), or by apolyalkyleneglycol radical of the formula —[O—(CH₂)_(r)]_(s)—NR(c)R(d),where r and s are, independently of each other an integer from 1 to 18,wherein any of the foregoing OH, SH, —CO—R(b), —NH—CO—NR(c)R(d),—NR(c)R(d), —NR(e)R(f), and —NR(e)R(g) or —NR(c)R(g) groups optionallycarries a protective group which is customary in nucleotide chemistry oris linked, where appropriate via a further linker, to one or more groupswhich favor intracellular uptake or serve for labeling a DNA or RNAprobe, or, when the oligonucleotide analog hybridizes to the targetnucleic acid, attack the latter while binding, cross-linking orcleaving, or (15) is a radical as defined under 3, 4 or 5 in which fromone to all the H atoms are substituted by halogen; R(a) is OH,(C₁-C₆)-alkoxy, (C₆-C₂₀)-aryloxy, NH₂ or NH-T, where T is analkylcarboxyl group or alkylamino group which is linked, optionally viaa further linker, to one or more groups which favor intracellular uptakeor serve as labeling for a DNA or RNA probe or, when the oligonucleotideanalog hybridizes to the target nucleic acid, attack the latter whilebinding, cross-linking or cleaving, R(b) is hydroxyl, (C₁-C₆)-alkoxy or—NR(c)R(d), R(c) and R(d) are, independently of each other, H or(C₁-C₆)-alkyl which is unsubstituted or substituted by —NR(e)R(f) or—NR(e)R(g), R(e) and R(f) are, independently of each other, H or(C₁-C₆)-alkyl, R(g) is (C₁-C₆)-alkyl-COOH; with the proviso that whenR¹⁵ and R¹⁶ are identical, they cannot be hydrogen, NO₂, NH₂, cyano orSiH₃; wherein functional groups are optionally protected with aprotective group which is customary in nucleotide chemistry, and thecurved bracket indicating that R^(2b) and the adjacent —Y^(b)R^(2b)radical can be located in the 2′ and 3′ position or else, conversely, inthe 3′ and 2′ position.
 2. The nucleotide monomer of the formula V ofclaim 1, wherein R¹⁵ is (1) NO₂, (2) NH₂, (3) —S—(C₁-C₆)-alkyl, (4)(C₁-C₆)-alkoxy, (5) (C₆-C₂₀)-aryloxy, (6) SiH₃,

(8) (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or (C₂-C₁₀)-alkynyl, any of whichmay be substituted by one or more radicals selected from the groupconsisting of SH, S—(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —NR(c)R(d),—CO—R(b), —NH—CO—NR(c)R(d), —NR(c)R(g), —NR(e)R(f) and —NR(e)R(g), or bya polyalkylene glycol radical of the formula—[O—(CH₂)_(r)]_(s)—NR(c)R(d), where r and a are, independently of eachother, an integer between 1 and 18, wherein any of the foregoing OH, SH,—CO—R(b), —NH—CO—NR(c)R(d), —NR(c)R(d), —NR(e)R(f), —NR(e)R(g) or—NR(c)R(g) groups optionally is linked, where appropriate via a furtherlinker, to one or more groups which favor intracellular uptake or serveas labeling for a DNA or RNA probe or, when the oligonucleotide analoghybridizes to the target nucleic acid, attack the latter while binding,cross-linking or cleaving, or (9) is (C₁-C₁₀)-alkyl, (C₂-C₁₀)-alkenyl or(C₂-C₁₀)-alkynyl in which from one to all the H atoms are substituted byhalogen; and R¹⁶ is hydrogen.
 3. The nucleotide monomer of formula V ofclaim 1, wherein R^(1b) is a succinyl linker, linked to anamino-functionalized or methylamino-functionalized solid support by anyamide or methylamide bond.
 4. The nucleotide monomer of formula V ofclaim 1, wherein at least one of R⁸ and R⁹ is isopropyl or ethyl.
 5. Acompound of the formula VI

in which, independently of each other, U′=U″=U′″ is hydroxyl ormercapto; e and f are 0 or 1; R¹³ is hydrogen, OH, C₁-C₁₈-alkoxy, orC₁-C₆-alkenyloxy; E and F are, independently of each other, H, OH orNH_(2;) and R¹⁵ and R¹⁶ are, independently of each other, (1) hydrogen,(2) halogen, (3) (C₁-C₁₀)-alkyl, (4) (C₂-C₁₀)-alkenyl, (5)(C₂-C₁₀)-alkynyl, (6) NO₂, (7) NH₂, (8) cyano, (9) —S—(C₁-C₆)-alkyl,(10) (C₁-C₆)-alkoxy, (11) (C₆-C₂₀)-aryloxy, (12) SiH₃,

(14) a radical as described under (3), (4) or (5) which is substitutedby one or more radicals selected from the group consisting of SH,S—(C₁-C₆)-alkyl, (C₁-C₆)-alkoxy, OH, —NR(c)R(d), —CO—R(b),—NH—CO—NR(c)R(d), —NR(c)R(g), —NR(e)R(f), and —NR(e)R(g), or by apolyalkyleneglycol radical of the formula —[O—(CH₂)_(r)]_(s)—NR(c)R(d),where r and s are, independently of each other an integer between 1 to18, wherein any of the foregoing OH, SH, —CO—R(b), —NH—CO—NR(c)R(d),—NR(c)R(d), —NR(e)R(f), and —NR(e)R(g) or —NR(c)R(g) groups optionallyis linked, where appropriate via a further linker, to one or more groupswhich favor intracellular uptake or serve as labeling for a DNA or RNAprobe or, when the oligonucleotide analog hybridizes to the targetnucleic acid, attack the latter while binding, cross-linking orcleaving, or are a radical as defined under (3), (4) or (5) in whichfrom one to all the H atoms are substituted by halogen; R(a) is OH,(C₁-C₆)-alkoxy, (C₆-C₂₀)-aryloxy, NH₂ or NH-T, with T representing analkylcarboxyl or alkylamino group which is linked, where appropriate viaa further linker, to one or more groups which favor intracellularuptake, or serve as labeling for a DNA or RNA probe or, when theoligonucleotide analog hybridizes to the target nucleic acid, attack thelatter while binding, cross-linking or cleaving, R(b) is hydroxyl,(C₁-C₆)-alkoxy or —NR(c)R(d), R(c) and R(d) are, independently of eachother, H or (C₁-C₆)-alkyl which is unsubstituted or substituted by—NR(e)R(f) or —NR(c)R(g) R(e) and R(f) are, independently of each other,H or (C₁-C₆)-alkyl, R(g) is (C₁-C₆)-alkyl-COOH, with the proviso thatwhen R¹⁵ and R¹⁶ are identical, they cannot be hydrogen, NO₂, NH₂, cyanoor SiH₃; with compounds of the formula VI being excepted in which R¹⁶ isH and R¹⁵ is (C₂-C₁₀)-alkynyl which is substituted by —NR(c)R(d) or—NR(e)R(f); and with the additional proviso that e and f are not O if Eis OH or NH₂, and F is OH, R¹⁶ is hydrogen and R¹⁵ is Br, Cl, F, cyano,(C₁-C₄)-alkyl, (C₂-C₄)-alkenyl or (C₂-C₄) -alkynyl.
 6. The compound offormula VI of claim 5, wherein at least one of R¹⁵ and R¹⁶ is a radicaldefined under (3), (4), or (5) in which from one to all of the H atomsis/are substituted by halogen, and said halogen is fluorine.
 7. Thenuclectide monomer of formula V of claim 1, wherein R^(1b) is asuccdinyl linker radical, bearing an activated ester.
 8. The nucleotidemonomer of formula V of claim 1, wherein at least one of R⁸ and R⁹ is


9. A polymerase chain reaction, wherein nucleotide triphosphatespolymerize and hybridize to template DNA to amplify desired sequences,and wherein one or more purine nucleotide triphosphates are replaced byone or more of the 7-deazapurine nucleotides of formula VI of claim 5,wherein e=f=1; and R¹³ is OH.
 10. The reaction of claim 9, wherein R¹⁶is H; and R¹⁸ is halogen.
 11. A method of sequencing polynucleotides,wherein modified purine nucleotide triphosphates are used as chainterminators or chain labeling agents, and wherein one or more purinenucleotide triphosphates are replaced by one or more of the7-deazapurine nucleotides of formula VI of claim 5, wherein e=f=1; andR¹³ is H or OH.